Novel RIP3 associated cell cycle proteins, compositions and methods of use

ABSTRACT

The present invention is directed to novel polypeptides, nucleic acids and related molecules which have an effect on or are related to the cell cycle. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Further provided by the present invention are methods for identifying novel compositions which mediate cell cycle bioactivity, and the use of such compositions in diagnosis and treatment of disease.

FIELD OF THE INVENTION

[0001] The present invention is directed to compositions involved incell cycle regulation and methods of use. More particularly, the presentinvention is directed to genes encoding proteins and proteins involvedin cell cycle regulation. Methods of use include use in assays screeningfor modulators of the cell cycle and use as therapeutics.

BACKGROUND OF THE INVENTION

[0002] Cells cycle through various stages of growth, starting with the Mphase, where mitosis and cytoplasmic division (cytokinesis) occurs. TheM phase is followed by the G1 phase, in which the cells resume a highrate of biosynthesis and growth. The S phase begins with DNA synthesis,and ends when the DNA content of the nucleus has doubled. The cell thenenters G2 phase, which ends when mitosis starts, signaled by theappearance of condensed chromosomes. Terminally differentiated cells arearrested in the G1 phase, and no longer undergo cell division.

[0003] The hallmark of a malignant cell is uncontrolled proliferation.This phenotype is acquired through the accumulation of gene mutations,the majority of which promote passage through the cell cycle. Cancercells ignore growth regulatory signals and remain committed to celldivision. Classic oncogenes, such as ras, lead to inappropriatetransition from G1 to S phase of the cell cycle, mimicking proliferativeextracellular signals. Cell cycle checkpoint controls ensure faithfulreplication and segregation of the genome. The loss of cell cyclecheckpoint control results in genomic instability, greatly acceleratingthe accumulation of mutations which drive malignant transformation.Thus, modulating cell cycle checkpoint pathways and other such pathwayswith therapeutic agents could exploit the differences between normal andtumor cells, both improving the selectivity of radio—and chemotherapy,and leading to novel cancer treatments. As another example, it would beuseful to control entry into apoptosis.

[0004] On the other hand, it is also sometimes desirable to enhanceproliferation of cells in a controlled manner. For example,proliferation of cells is useful in wound healing and where growth oftissue is desirable. Thus, identifying modulators which promote, enhanceor deter the inhibition of proliferation is desirable.

[0005] Despite the desirability of identifying cell cycle components andmodulators, there is a deficit in the field of such compounds.Accordingly, it would be advantageous to provide compositions andmethods useful in screening for modulators of the cell cycle. It wouldalso be advantageous to provide novel compositions which are involved inthe cell cycle.

SUMMARY OF THE INVENTION

[0006] The present invention provides cell cycle proteins and nucleicacids which encode such proteins. Also provided are methods forscreening for a bioactive agent capable of modulating the cell cycle.The method comprises combining a cell cycle protein and a candidatebioactive agent and a cell or a population of cells, and determining theeffect on the cell in the presence and absence of the candidate agent.Therapeutics for regulating or modulating the cell cycle are alsoprovided.

[0007] In one aspect, a recombinant nucleic acid encoding a cell cycleprotein of the present invention comprises a nucleic acid thathybridizes under high stringency conditions to a sequence complementaryto that set forth in FIGS. 1 or 3. In a preferred embodiment, the cellcycle protein provided herein binds to RIP3.

[0008] In one embodiment, a recombinant nucleic acid is provided whichcomprises a nucleic acid sequence as set forth in FIGS. 1 or 3. Inanother embodiment, a recombinant nucleic acid encoding a cell cycleprotein is provided which comprises a nucleic acid sequence having atleast 85% sequence identity to a sequence as set forth in FIGS. 1 or 3.In a further embodiment, provided herein is a recombinant nucleic acidencoding an amino acid sequence as depicted in FIG. 2.

[0009] In another aspect of the invention, expression vectors areprovided. The expression vectors comprise one or more of the recombinantnucleic acids provided herein operably linked to regulatory sequencesrecognized by a host cell transformed with the nucleic acid. Furtherprovided herein are host cells comprising the vectors and recombinantnucleic acids provided herein. Moreover, provided herein are processesfor producing a cell cycle protein comprising culturing a host cell asdescribed herein under conditions suitable for expression of the cellcycle protein. In one embodiment, the process includes recovering thecell cycle protein.

[0010] Also provided herein are recombinant cell cycle proteins encodedby the nucleic acids of the present invention. In one aspect, arecombinant polypeptide is provided herein which comprises an amino acidsequence having at least 80% sequence identity with a sequence as setforth in FIGS. 2 or 4. In one embodiment, a recombinant cell cycleprotein is provided which comprises an amino acid sequence as set forthin FIGS. 2 or 4.

[0011] In another aspect, the present invention provides isolatedpolypeptides which specifically bind to a cell cycle protein asdescribed herein. Examples of such isolated polypeptides includeantibodies. Such an antibody can be a monoclonal antibody. In oneembodiment, such an antibody reduces or eliminates the biologicalfunction of said cell cycle protein.

[0012] Further provided herein are methods for screening for a bioactiveagent capable of binding to a cell cycle protein. In one embodiment themethod comprises combining a cell cycle protein and a candidatebioactive agent, and determining the binding of said candidate bioactiveagent to said cell cycle protein.

[0013] In another aspect, provided herein is a method for screening fora bioactive agent capable of interfering with the binding of a cellcycle protein and a RIP3 protein. In one embodiment, such a methodcomprises combining a cell cycle protein, a candidate bioactive agentand a RIP3 protein, and determining the binding of the cell cycleprotein and the RIP3 protein. If desired, the cell cycle protein and theRIP3 protein can be combined first.

[0014] Further provided herein are methods for screening for a bioactiveagent capable of modulating the activity of cell cycle protein. In oneembodiment the method comprises adding a candidate bioactive agent to acell comprising a recombinant nucleic acid encoding a cell cycleprotein, and determining the effect of the candidate bioactve agent onthe cell. In a preferred embodiment, a library of candidate bioactiveagents is added to a plurality of cells comprising a recombinant nucleicacid encoding a cell cycle protein.

[0015] Other aspects of the invention will become apparent to theskilled artisan by the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the nucleic acid sequence encoding cell cycle proteinSRIK-isoform 1, SEQ ID NO: 1. A translation start codon (ATG), anupstream termination codon (TAG at position 528), and a translationtermination codon (TAG at position 1467) are in bold and underlined.

[0017]FIG. 2 shows the amino acid sequence of cell cycle proteinSRIK-isoform 1, SEQ ID NO: 2.

[0018]FIG. 3 shows the nucleic acid sequence encoding cell cycle proteinSRIK-isoform 2, SEQ ID NO: 3. A translation start codon (ATG), anupstream termination codon (TAG at position 528), and a translationtermination codon (TAG at position 1468) are in bold and underlined.

[0019]FIG. 4 shows expression of the SRIK protein in various mammaliantissues and cell lines.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides cell cycle proteins and nucleicacids which encode such proteins. Also provided are methods forscreening for a bioactive agent capable of modulating the cell cycle.The method comprises combining a cell cycle protein and a candidatebioactive agent and a cell or a population of cells, and determining theeffect on the cell in the presence and absence of the candidate agent.Other screening assays including binding assays are also provided hereinas described below. Therapeutics for regulating or modulating the cellcycle are also provided and described herein. Diagnostics, as furtherdescribed below, are also provided herein.

[0021] A cell cycle protein of the present invention may be identifiedin several ways. “Protein” in this sense includes proteins,polypeptides, and peptides. The cell cycle proteins of the inventionfall into two general classes: proteins that are completely novel, i.e.are not part of a public database as of the time of discovery, althoughthey may have homology to either known proteins or peptides encoded byexpressed sequence tags (ESTs). Alternatively, the cell cycle proteinsare known proteins, but that were not known to be involved in the cellcycle; i.e. they are identified herein as having a novel biologicalfunction. Accordingly, a cell cycle protein may be initially identifiedby its association with a protein known to be involved in the cellcycle. Wherein the cell cycle proteins and nucleic acids are novel,compositions and methods of use are provided herein. In the case thatthe cell cycle proteins and nucleic acids were known but not known to beinvolved in cell cycle activity as described herein, methods of use,i.e. functional screens, are provided.

[0022] In one embodiment provided herein, a cell cycle protein asdefined herein has one or more of the following characteristics: bindingto RIP3; homology to serine/threonine protein kinases; and cell cycleprotein activity as described herein. RIP3 and associated processes arefurther discussed below. In one aspect, the homology to serine/threonineprotein kinases is found, e.g., using the BLAST search program inGenBank [Altschul et al., Nuceic Acids Res. 25: 3389-3402 (1998)]. Moreparticularly, in one embodyment the following parameters were used toidentify sequences having homology to SRIK-isoform 1 herein: Database:non-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR;Lambda of 0.318, K of 0.136, H of 0.0; Gapped Lambda of 0.27, K of0.047, H of 4.94e-324; Matrix: BLOSUM62; Gap penalties: Existence: 11,Extension: 1.

[0023] In one embodiment, the cell cycle protein is termed SRIK. Thecharacteristics described below can apply to any of the cell cycleproteins provided herein, however, SRIK is used for illustrativepurposes. SRIK is similar to proteins having a serine/threonine kinasedomain. Preferably, SRIK binds to RIP3, a kinase which is involved withtumor necrosis factor receptor (TNFR) signaling proteins [see Yu et al.,Curr. Biol. 9(10):539-542 (1999); Sun et al., J. Biol. Chem. 274(24):16871-16875 (1999)]. Over-expression of RIP3 has been reported to induceapoptosis [Sun et al., supra], and expression of the C-terminal (lackingthe kinase domain) in mammalian cells also induced apoptosis andactivated the transcription factor NF-K B [Yu et al., supra ].

[0024] RIP3 binds RIP (receptor-interacting protein), which is acomponent of the signaling complexes recruited and assembled in responseto stimulation of the TNFR's. The TNFR superfamily is involved in theinduction of cellular signals resulting in cell growth, differentiation,and cell death. RIP is recruited by TRADD to TNFR1 in a TNF-dependentprocess. RIP and homologous TNFR1-associated kinase RIP2 have beenreported to induce both NF-K B activation and apoptosis [Hsu et al.,supra; McCarthy et al., J. Biol. Chem. 273(27):16968-16975 (1998); Thomeet al., Curr. Biol. 8(15): 885-888(1998)]. RIP is reported to mediateacfivation of NF-K B that results from TNF stimulation of TNFR1 [seeKelliher et al., Immunity 8(3):297-303 (1998); Hsu et al., Immunity4(4):387-396 (1996)]. RIP3 has been reported to attenuate both directRIP and stimulated TNFR1 activation of NF-K B [Sun et al., supra],consistent with RIP3's role as a mediator of TNFR1 signal transduction.RIP is also associated with other TNFR's [see, e.g., Chaudhary et al.,Immunity 7(6): 821-830 (1997)].

[0025] Native SRIK is predominantly expressed in T-cells, hela cells andleukocytes. Leukocytes are known be involved in localized inflammatoryprocesses, accumulating at sites of inflammation [see Walcheck et al.,Nature 380(6576): 720-723 (1996); Hartwig et al., J. Appl. Physiol.87(2): 743-749 (1999)]. In fact, most treatments for inflammatory jointdisease are directed to inhibition of the leukocyte infiltration andaccumulation during inflammation [Parnham, Biochem. Pharmacol. 58(2):209-215 (1999)]. NF-K B is thought to be a key player in inflammationdisease, implicating the above processes in inflammation diseases aspart their involvement in cell cycle signaling.

[0026] In one embodiment, cell cycle nucleic acids or cell cycleproteins are initially identified by substantial nucleic acid and/oramino acid sequence identity or similarity to the sequence(s) providedherein. In a preferred embodiment, cell cycle nucleic acids or cellcycle proteins have sequence identity or similarity to the sequencesprovided herein as described below and one or more of the cell cycleprotein bioactivities as further described below. Such sequence identityor similarity can be based upon the overall nucleic acid or amino acidsequence.

[0027] In a preferred embodiment, a protein is a “cell cycle protein” asdefined herein if the overall sequence identity of the amino acidsequence of FIG. 2 is preferably greater than about 75%, more preferablygreater than about 80%, even more preferably greater than about 85% andmost preferably greater than 90%. In some embodiments the sequenceidentity will be as high as about 93 to 95 or 98%.

[0028] In another preferred embodiment, a cell cycle protein has anoverall sequence similarity with the amino acid sequence of FIG. 2greater than about 80%, more preferably greater than about 85%, evenmore preferably greater than about 90% and most preferably greater than93%. In some embodiments the sequence identity will be as high as about95 to 98 or 99%.

[0029] As is known in the art, a number of different programs can beused to identify whether a protein (or nucleic acid as discussed below)has sequence identity or similarity to a known sequence. Sequenceidentity and/or similarity is determined using standard techniques knownin the art, including, but not limited to, the local sequence identityalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thesequence identity alignment algorithm of Needleman & Wunsch, J. Mol.Biool. 48:443 (1970), by the search for similarity method of Pearson &Lipman, PNAS USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal, Nucl. Acid Res. 12:387-395 (1984), preferably using the defaultsettings, or by inspection. Preferably, percent identity is calculatedby FastDB based upon the following parameters: mismatch penalty of 1;gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30,“Current Methods in Sequence Comparison and Analysis,” MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp 127-149(1988), Alan R. Liss, Inc.

[0030] An example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments. It can also plot a tree showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987); the method is similar to that describedby Higgins & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parametersincluding a default gap weight of 3.00, a default gap length weight of0.10, and weighted end gaps.

[0031] Another example of a useful algorithm is the BLAST algorithm,described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) andKarlin et al., PNAS USA 90:5873-5787 (1993). A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., Methods in EnzVmologv, 266: 460-480 (1996);http://blast.wustl/edu/blast/ README.html]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

[0032] An additional useful algorithm is gapped BLAST as reported byAltschul et al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST usesBLOSUM-62 substitution scores; threshold T parameter set to 9; thetwo-hit method to trigger ungapped extensions; charges gap lengths of ka cost of 10+k; X_(u) set to 16, and X_(g) set to 40 for database searchstage and to 67 for the output stage of the algorithms. Gappedalignments are triggered by a score corresponding to ˜22 bits.

[0033] A % amino acid sequence identity value is determined by thenumber of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

[0034] In a similar manner, “percent (%) nucleic acid sequence identity”with respect to the coding sequence of the polypeptides identifiedherein is defined as the percentage of nucleotide residues in acandidate sequence that are identical with the nucleotide residues inthe coding sequence of the cell cycle protein. A preferred methodutilizes the BLASTN module of WU-BLAST-2 set to the default parameters,with overlap span and overlap fraction set to 1 and 0.125, respectively.

[0035] The alignment may include the introduction of gaps in thesequences to be aligned. In addition, for sequences which contain eithermore or fewer amino acids than the protein encoded by the sequences inFIGS. 1 or 3, it is understood that in one embodiment, the percentage ofsequence identity will be determined based on the number of identicalamino acids in relation to the total number of amino acids. Thus, forexample, sequence identity of sequences shorter than that shown in FIG.2, as discussed below, will be determined using the number of aminoacids in the shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

[0036] In one embodiment, only identities are scored positively (+1) andall forms of sequence variation including gaps are assigned a value of“0”, which obviates the need for a weighted scale or parameters asdescribed below for sequence similarity calculations. Percent sequenceidentity can be calculated, for example, by dividing the number ofmatching identical residues by the total number of residues of the“shorter” sequence in the aligned region and multiplying by 100. The“longer” sequence is the one having the most actual residues in thealigned region.

[0037] As will be appreciated by those skilled in the art, the sequencesof the present invention may contain sequencing errors. That is, theremay be incorrect nucleosides, frameshifts, unknown nucleosides, or othertypes of sequencing errors in any of the sequences; however, the correctsequences will fall within the homology and stringency definitionsherein.

[0038] Cell cycle proteins of the present invention may be shorter orlonger than the amino acid sequence encoded by the nucleic acid shown inFIGS. 1 or 3. Thus, in a preferred embodiment, included within thedefinition of cell cycle proteins are portions or fragments of the aminoacid sequence encoded by the nucleic acid sequence provided herein. Inone embodiment herein, fragments of cell cycle proteins are consideredcell cycle proteins if a) they share at least one antigenic epitope; b)have at least the indicated sequence identity; c) and preferably havecell cycle biological activity as further defined herein. In some cases,where the sequence is used diagnostically, that is, when the presence orabsence of cell cycle protein nucleic acid is determined, only theindicated sequence identity is required. The nucleic acids of thepresent inventon may also be shorter or longer than the sequence inFIGS. 1 or 3. The nucleic acid fragments include any portion of thenucleic acids provided herein which have a sequence not exactlypreviously identified; fragments having sequences with the indicatedsequence identity to that portion not previously identified are providedin an embodiment herein.

[0039] In addition, as is more fully outlined below, cell cycle proteinscan be made that are longer than those depicted in FIG. 2; for example,by the addition of epitope or purification tags, the addition of otherfusion sequences, or the elucidation of additional coding and non-codingsequences. As described below, the fusion of a cell cycle peptide to afluorescent peptide, such as Green Fluorescent Peptide (GFP), isparticularly preferred.

[0040] Cell cycle proteins may also be identified as encoded by cellcycle nucleic acids which hybridize to the sequence depicted in FIGS. 1or 3, the complement thereof, as outlined herein. Hybridizationconditions are further described below.

[0041] In a preferred embodiment, when a cell cycle protein is to beused to generate antibodies, a cell cycle protein must share at leastone epitope or determinant with the full length protein. By “epitope” or“determinant” herein is meant a portion of a protein which will generateand/or bind an antibody. Thus, in most instances, antibodies made to asmaller cell cycle protein will be able to bind to the full lengthprotein. In a preferred embodiment, the epitope is unique; that is,antibodies generated to a unique epitope show little or nocross-reactivity. The term “antibody” includes antibody fragments, asare known in the art, including Fab Fab₂, single chain antibodies (Fvfor example), chimeric antibodies, etc., either produced by themodification of whole antibodies or those synthesized de novo usingrecombinant DNA technologies.

[0042] In a preferred. embodiment, the antibodies to a cell cycleprotein are capable of reducing or eliminating the biological functionof the cell cycle proteins described herein, as is described below. Thatis, the addition of anti-cell cycle protein antibodies (eitherpolyclonal or preferably monoclonal) to cell cycle proteins (or cellscontaining cell cycle proteins) may reduce or eliminate the cell cycleactivity. Generally, at least a 25% decrease in activity is preferred,with at least about 50% being particularly preferred and about a 95-100%decrease being especially preferred.

[0043] The cell cycle antibodies of the invention specifically bind tocell cycle proteins. In a preferred embodiment, the antibodiesspecifically bind to cell cycle proteins. By “specifically bind” hereinis meant that the antibodies bind to the protein with a binding constantin the range of at least 10⁻⁴-10⁻⁶M⁻¹, with a preferred range being10⁻⁷-10⁻¹M⁻¹. Antibodies are further described below.

[0044] In the case of the nucleic acid, the overall sequence identity ofthe nucleic acid sequence is commensurate with amino acid sequenceidentity but takes into account the degeneracy in the genetic code andcodon bias of different organisms. Accordingly, the nucleic acidsequence identity may be either lower or higher than that of the proteinsequence. Thus the sequence identity of the nucleic acid sequence ascompared to the nucleic acid sequence of the Figure is preferablygreater than 75%, more preferably greater than about 80%, particularlygreater than about 85% and most preferably greater than 90%. In someembodiments the sequence identity will be as high as about 93 to 95 or98%.

[0045] In a preferred embodiment, a cell cycle nucleic acid encodes acell cycle protein. As will be appreciated by those in the art, due tothe degeneracy of the genetic code, an extremely large number of nucleicacids may be made, all of which encode the cell cycle proteins of thepresent invention. Thus, having identified a particular amino acidsequence, those skilled in the art could make any number of differentnucleic acids, by simply modifying the sequence of one or more codons ina way which does not change the amino acid sequence of the cell cycleprotein.

[0046] In one embodiment, the nucleic acid is determined throughhybridization studies. Thus, for example, nucleic acids which hybridizeunder high stringency to the nucleic acid sequence shown in FIGS. 1 or3, or its complement is considered a cell cycle nucleic acid. Highstringency conditions are known in the art; see for example Maniatis etal., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al., both of which arehereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength pH. The T_(m) is the temperature (under defined ionic strength,pH and nucleic acid concentration) at which 50% of the probescomplementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g. greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide.

[0047] In another embodiment, less stringent hybridization conditionsare used; for example, moderate or low stringency conditions may beused, as are known in the art; see Maniatis and Ausubel, supra, andTijssen, supra.

[0048] The cell cycle proteins and nucleic acids of the presentinvention are preferably recombinant. As used herein and further definedbelow, “nucleic acid” may refer to either DNA or RNA, or molecules whichcontain both deoxy—and ribonucleotides. The nucleic acids includegenomic DNA, cDNA and oligonucleotides including sense and anti-sensenucleic acids. Such nucleic acids may also contain modifications in theribose-phosphate backbone to increase stability and half life of suchmolecules in physiological environments.

[0049] The nucleic acid may be double stranded, single stranded, orcontain portions of both double stranded or single stranded sequence. Aswill be appreciated by those in the art, the depiction of a singlestrand (“Watson”) also defines the sequence of the other strand(“Crick”); thus the sequences depicted in the Figures also include thecomplement of the sequence. By the term “recombinant nucleic acid”herein is meant nucleic acid, originally formed in vitro, in general, bythe manipulation of nucleic acid by endonucleases, in a form notnormally found in nature. Thus an isolated cell cycle nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e. using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention.

[0050] Similarly, a “recombinant protein” is a protein made usingrecombinant techniques, i.e. through the expression of a recombinantnucleic acid as depicted above. A recombinant protein is distinguishedfrom naturally occurring protein by at least one or morecharacteristics. For example, the protein may be isolated or purifiedaway from some or all of the proteins and compounds with which it isnormally associated in its wild type host, and thus may be substantiallypure. For example, an isolated protein is unaccompanied by at least someof the material with which it is normally associated in its naturalstate, preferably constituting at least about 0.5%, more preferably atleast about 5% by weight of the total protein in a given sample. Asubstantially pure protein comprises at least about 75% by weight of thetotal protein, with at least about 80% being preferred, and at leastabout 90% being particularly preferred. The definition includes theproduction of a cell cycle protein from one organism in a differentorganism or host cell. Alternatively, the protein may be made at asignificantly higher concentration than is normally seen, through theuse of a inducible promoter or high expression promoter, such that theprotein is made at increased concentration levels. Alternatively, theprotein may be in a form not normally found in nature, as in theaddition of an epitope tag or amino acid substitutions, insertions anddeletions, as discussed below.

[0051] In one embodiment, the present invention provides cell cycleprotein variants. These variants fall into one or more of three classes:substitutional, insertional or deletonal variants. These variantsordinarily are prepared by site specific mutagenesis of nucleotides inthe DNA encoding a cell cycle protein, using cassette or PCR mutagenesisor other techniques well known in the art, to produce DNA encoding thevariant, and thereafter expressing the DNA in recombinant cell cultureas outlined above. However, variant cell cycle protein fragments havingup to about 100-150 residues may be prepared by in vitro synthesis usingestablished techniques. Amino acid sequence variants are characterizedby the predetermined nature of the variation, a feature that sets themapart from naturally occurring allelic or interspecies variation of thecell cycle protein amino acid sequence. The variants typically exhibitthe same qualitative biological activity as the naturally occurringanalogue, although variants can also be selected which have modifiedcharacteristics as will be more fully outlined below.

[0052] While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed cell cycle variants screenedfor the optimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays of cell cycleprotein activities.

[0053] Amino acid substitutions are typically of single residues;insertions usually will be on the order of from about 1 to 20 aminoacids, although considerably larger insertions may be tolerated.Deletions range from about 1 to about 20 residues, although in somecases deletions may be much larger.

[0054] Substitutions, deletions, insertions or any combination thereofmay be used to arrive at a final derivative. Generally these changes aredone on a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the cell cycle protein aredesired, substitutions are generally made in accordance with thefollowing chart: CHART I Original Residue Exemplary Substitutions AlaSer Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro HisAsn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile PheMet, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[0055] Substantial changes in function or immunological identity aremade by selecting substitutions that are less conservative than thoseshown in Chart I. For example, substitutions may be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example the alpha-helical or beta-sheetstructure; the charge or hydrophobicity of the molecule at the targetsite; or the bulk of the side chain. The substitutions which in generalare expected to produce the greatest changes in the polypeptide'sproperties are those in which (a) a hydrophilic residue, e.g. seryl orthreonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl,isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline issubstituted for (or by) any other residue; (c) a residue having anelectropositive side chain, e.g. lysyl, arginyl, or histidyl, issubstituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine.

[0056] The variants typically exhibit the same qualitative biologicalactivity and will elicit the same immune response as thenaturally-occurring analogue, although variants also are selected tomodify the characteristics of the cell cycle proteins as needed.Alternatively, the variant may be designed such that the biologicalactivity of the cell cycle protein is altered. For example,glycosylation sites may be altered or removed.

[0057] Covalent modifications of cell cycle polypeptides are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of a cell cyclepolypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N-or C-terminal residues of acell cycle polypeptide. Derivatization with bifunctional agents isuseful, for instance, for crosslinking cell cycle to a water-insolublesupport matrix or surface for use in the method for purifying anti-cellcycle antibodies or screening assays, as is more fully described below.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctonal imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidyl-propionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

[0058] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the “-amino groups of lysine, arginine, and histidineside chains [T.E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0059] Another type of covalent modification of the cell cyclepolypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequencecell cycle polypeptide, and/or adding one or more glycosylation sitesthat are not present in the native sequence cell cycle polypeptide.

[0060] Addition of glycosylation sites to cell cycle polypeptides may beaccomplished by altering the amino acid sequence thereof. The alterationmay be made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the native sequence cell cyclepolypeptide (for O-linked glycosylation sites). The cell cycle aminoacid sequence may optionally be altered through changes at the DNAlevel, particularly by mutating the DNA encoding the cell cyclepolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

[0061] Another means of increasing the number of carbohydrate moietieson the cell cycle polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0062] Removal of carbohydrate moieties present on the cell cyclepolypeptide may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo-andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

[0063] Another type of covalent modification of cell cycle compriseslinking the cell cycle polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0064] Cell cycle polypeptides of the present invention may also bemodified in a way to form chimeric molecules comprising a cell cyclepolypeptide fused to another, heterologous polypeptide or amino acidsequence. In one embodiment, such a chimeric molecule comprises a fusionof a cell cycle polypeptide with a tag polypeptide which provides anepitope to which an anti-tag antibody can selectively bind. The epitopetag is generally placed at the amino-or carboxyl-terminus of the cellcycle polypeptide. The presence of such epitope-tagged forms of a cellcycle polypeptide can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables the cell cyclepolypeptide to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. In an alternative embodiment, the chimeric molecule maycomprise a fusion of a cell cycle polypeptide with an immunoglobulin ora particular region of an immunoglobulin. For a bivalent form of thechimeric molecule, such a fusion could be to the Fc region of an IgGmolecule as discussed further below.

[0065] Various tag polypeptides and their respective antibodies are wellknown in the art. Examples include poly-histidine (poly-his) orpoly-histidineglycine (poly-hisgly) tags; the flu HA tag polypeptide andits antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)];the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto[Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; andthe Herpes Simplex virus glycoprotein D (gD) tag and its antibody[Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tagpolypeptides include the Flag-peptide [Hopp et al., BioTechnology,6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science,255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol.Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)].

[0066] In an embodiment herein, cell cycle proteins of the cell cyclefamily and cell cycle proteins from other organisms are cloned andexpressed as outlined below. Thus, probe or degenerate polymerase chainreaction (PCR) primer sequences may be used to find other related cellcycle proteins from humans or other organisms. As will be appreciated bythose in the art, particularly useful probe and/or PCR primer sequencesinclude the unique areas of the cell cycle nucleic acid sequence. As isgenerally known in the art, preferred PCR primers are from about 15 toabout 35 nucleotides in length, with from about 20 to about 30 beingpreferred, and may contain inosine as needed. The conditions for the PCRreaction are well known in the art. It is therefore also understood thatprovided along with the sequences in the sequences listed herein areportions of those sequences, wherein unique portions of 15 nucleotidesor more are particularly preferred. The skilled artisan can routinelysynthesize or cut a nucleotide sequence to the desired length.

[0067] Once isolated from its natural source, e.g., contained within aplasmid or other vector or excised therefrom as a linear nucleic acidsegment, the recombinant cell cycle nucleic acid can be further-used asa probe to identify and isolate other cell cycle nucleic acids. It canalso be used as a “precursor” nucleic acid to make modified or variantcell cycle nucleic acids and proteins.

[0068] Using the nucleic acids of the present invention which encode acell cycle protein, a variety of expression vectors are made. Theexpression vectors may be either self-replicating extrachromosomalvectors or vectors which integrate into a host genome. Generally, theseexpression vectors include transcriptional and translational regulatorynucleic acid operably linked to the nucleic acid encoding the cell cycleprotein. The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to ublize promoters,polyadenylation signals, and enhancers.

[0069] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypepbde if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. As another example, operablylinked refers to DNA sequences linked so as to be contiguous, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the cell cycle protein; for example, transcriptional andtranslational regulatory nucleic acid sequences from Bacillus arepreferably used to express the cell cycle protein in Bacillus. Numeroustypes of appropriate expression vectors, and suitable regulatorysequences are known in the art for a variety of host cells.

[0070] In general, the transcriptional and translational regulatorysequences may include, but are not limited to, promoter sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, and enhancer or activatorsequences. In a preferred embodiment, the regulatory sequences include apromoter and transcriptional start and stop sequences.

[0071] Promoter sequences encode either constitutive or induciblepromoters. The promoters may be either naturally occurring promoters orhybrid promoters. Hybrid promoters, which combine elements of more thanone promoter, are also known in the art, and are useful in the presentinvention.

[0072] In addition, the expression vector may comprise additionalelements. For example, the expression vector may have two replicationsystems, thus allowing it to be maintained in two organisms, for examplein mammalian or insect cells for expression and in a procaryotic hostfor cloning and amplification. Furthermore, for integrating expressionvectors, the expression vector contains at least one sequence homologousto the host cell genome, and preferably two homologous sequences whichflank the expression construct. The integrating vector may be directedto a specific locus in the host cell by selecting the appropriatehomologous sequence for inclusion in the vector. Constructs forintegrating vectors are well known in the art.

[0073] In addition, in a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selection genes are well known in the art and will vary withthe host cell used.

[0074] A preferred expression vector system is a retroviral vectorsystem such as is generally described in PCT/US97/01019 andPCT/US97/01048, both of which are hereby expressly incorporated byreference.

[0075] Cell cycle proteins of the present invention are produced byculturing a host cell transformed with an expression vector containingnucleic acid encoding a cell cycle protein, under the appropriateconditions to induce or cause expression of the cell cycle protein. Theconditions appropriate for cell cycle protein expression will vary withthe choice of the expression vector and the host cell, and will beeasily ascertained by one skilled in the art through routineexperimentation. For example, the use of constitutive promoters in theexpression vector will require optimizing the growth and proliferationof the host cell, while the use of an inducible promoter requires theappropriate growth conditions for induction. In addition, in someembodiments, the timing of the harvest is important. For example, thebaculoviral systems used in insect cell expression are lytic viruses,and thus harvest time selection can be crucial for product yield.

[0076] Appropriate host cells include yeast, bacteria, archebacteria,fungi, and insect and animal cells, including mammalian cells. Ofparticular interest are Drosophila melangaster cells, Saccharomycescerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells,fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid andlymphoid cell lines, tumor cell lines, spleen cells, thymus cells, smallintestine cells, colon cells, B cell lines, T cell lines and peripheralblood leukocyte cells.

[0077] In a preferred embodiment, the cell cycle proteins are expressedin mammalian cells. Mammalian expression systems are also known in theart, and include retroviral systems. A mammalian promoter is any DNAsequence capable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for cell cycleprotein into mRNA. A promoter will have a transcription initiatingregion, which is usually placed proximal to the 5′ end of the codingsequence, and a TATA box, using a located 25-30 base pairs upstream ofthe transcription initiation site. The TATA box is thought to direct RNApolymerase II to begin RNA synthesis at the correct site. A mammalianpromoter will also contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box. An upstream promoter element determines the rate at whichtranscription is initiated and can act in either orientation. Ofparticular use as mammalian promoters are the promoters from mammalianviral genes, since the viral genes are often highly expressed and have abroad host range. Examples include the SV40 early promoter, mousemammary tumor virus LTR promoter, adenovirus major late promoter, herpessimplex virus promoter, and the CMV promoter.

[0078] Typically, transcription termination and polyadenylationsequences recognized by mammalian cells are regulatory regions located3′ to the translation stop codon and thus, together with the promoterelements, flank the coding sequence. The 3′ terminus of the mature mRNAis formed by site-specific post-translational cleavage andpolyadenylation. Examples of transcription terminator and polyadenlytionsignals include those derived form SV40.

[0079] The methods of introducing exogenous nucleic acid into mammalianhosts, as well as other hosts, is well known in the art, and will varywith the host cell used. Techniques include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, viral infection,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei.

[0080] In a preferred embodiment, cell cycle proteins are expressed inbacterial systems. Bacterial expression systems are well known in theart.

[0081] A suitable bacterial promoter is any nucleic acid sequencecapable of binding bacterial RNA polymerase and initiating thedownstream (3′) transcription of the coding sequence of cell cycleprotein into mRNA. A bacterial promoter has a transcription initiationregion which is usually placed proximal to the 5′ end of the codingsequence. This transcription initiation region typically includes an RNApolymerase binding site and a transcription initiation site. Sequencesencoding metabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

[0082] In addition to a functioning promoter sequence, an efficientribosome binding site is desirable. In E. coli, the ribosome bindingsite is called the Shine-Delgarno (SD) sequence and includes aninitition codon and a sequence 3-9 nucleotides in length located 3 -11nucleotides upstream of the initiation codon.

[0083] The expression vector may also include a signal peptide sequencethat provides for secretion of the cell cycle protein in bacteria. Thesignal sequence typically encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell, as is well known in the art. The protein is either secretedinto the growth media (gram-positive bacteria) or into the periplasmicspace, located between the inner and outer membrane of the cell(gram-negative bacteria).

[0084] The bacterial expression vector may also include a selectablemarker gene to allow for the selection of bacterial strains that havebeen transformed. Suitable selection genes include genes which renderthe bacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

[0085] These components are assembled into expression vectors.Expression vectors for bacteria are well known in the art, and includevectors for Bacillus subtilis, E. coli, Streptococcus cremoris, andStreptococcus lividans, among others.

[0086] The bacterial expression vectors are transformed into bacterialhost cells using techniques well known in the art, such as calciumchloride treatment, electroporation, and others.

[0087] In one embodiment, cell cycle proteins are produced in insectcells. Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

[0088] In a preferred embodiment, cell cycle protein is produced inyeast cells. Yeast expression systems are well known in the art, andinclude expression vectors for Saccharomyces cerevisiae, Candidaalbicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilisand K. lactis, Pichia guillerimondii and P. pastoris,Schizosaccharomyces pombe, and Yarrowia lipolytica. Preferred promotersequences for expression in yeast include the inducible GAL1,10promoter, the promoters from alcohol dehydrogenase, enolase,glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

[0089] The cell cycle protein may also be made as a fusion protein,using techniques well known in the art. Thus, for example, for thecreation of monoclonal antibodies, if the desired epitope is small, thecell cycle protein may be fused to a carrier protein to form animmunogen. Alternatively, the cell cycle protein may be made as a fusionprotein to increase expression, or for other reasons. For example, whenthe cell cycle protein is a cell cycle peptide, the nucleic acidencoding the peptide may be linked to other nucleic acid for expressionpurposes. Similarly, cell cycle proteins of the invention can be linkedto protein labels, such as green fluorescent protein (GFP), redfluorescent protein (RFP), blue fluorescent protein (BFP), yellowfluorescent protein (YFP), etc.

[0090] In one embodiment, the cell cycle nucleic acids, proteins andantibodies of the invention are labeled. By “labeled” herein is meantthat a compound has at least one element, isotope or chemical compoundattached to enable the detection of the compound. In general, labelsfall into three classes: a) isotopic labels, which may be radioactive orheavy isotopes; b) immune labels, which may be antibodies or antigens;and c) colored or fluorescent dyes. The labels may be incorporated intothe compound at any position.

[0091] In a preferred embodiment, the cell cycle protein is purified orisolated after expression. Cell cycle proteins may be isolated orpurified in a variety of ways known to those skilled in the artdepending on what other components are present in the sample. Standardpurification methods include electrophoretic, molecular, immunologicaland chromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography, and chromatofocusing.For example, the cell cycle protein may be purified using a standardanti-cell cycle antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.For general guidance in suitable purification techniques, see Scopes,R., Protein Purification, Springer-Verlag, NY (1982). The degree ofpurification necessary will vary depending on the use of the cell cycleprotein. In some instances no purification will be necessary.

[0092] Once expressed and purified if necessary, the cell cycle proteinsand nucleic acids are useful in a number of applications.

[0093] The nucleotide sequences (or their complement) encoding cellcycle proteins have various applications in the art of molecularbiology, including uses as hybridization probes, in chromosome and genemapping and in the generation of anti-sense RNA and DNA. Cell cycleprotein nucleic acid will also be useful for the preparation of cellcycle proteins by the recombinant techniques described herein.

[0094] The full-length native sequence cell cycle protein gene, orportions thereof, may be used as hybridization probes for a cDNA libraryto isolate other genes (for instance, those encoding naturally-occurringvariants of cell cycle protein or cell cycle protein from other species)which have a desired sequence identity to the cell cycle protein codingsequence. Optionally, the length of the probes will be about 20 to about50 bases. The hybridization probes may be derived from the nucleotidesequences herein or from genomic sequences including promoters, enhancerelements and introns of native sequences as provided herein. By way ofexample, a screening method will comprise isolating the coding region ofthe cell cycle protein gene using the known DNA sequence to synthesize aselected probe of about 40 bases. Hybridization probes may be labeled bya variety of labels, including radionucleotides such as ³²P or ³⁵S, orenzymatic labels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the cell cycle protein gene of the presentinvention can be used to screen libraries of human cDNA, genoric DNA ormRNA to determine which members of such libraries the probe hybridizes.

[0095] Nucleotide sequences encoding a cell cycle protein can also beused to construct hybridization probes for mapping the gene whichencodes that cell cycle protein and for the genetic analysis ofindividuals with genetic disorders. The nucleotide sequences providedherein may be mapped to a chromosome and specific regions of achromosome using known techniques, such as in situ hybridization,linkage analysis against known chromosomal markers, and hybridizationscreening with libraries.

[0096] Nucleic acids which encode cell cycle protein or its modifiedforms can also be used to generate either transgenic animals or “knockout” animals which, in turn, are useful in the development and screeningof therapeutically useful reagents. A transgenic animal (e.g., a mouseor rat) is an animal having cells that contain a transgene, whichtransgene was introduced into the animal or an ancestor of the animal ata prenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding a cell cycle protein can beused to clone genomic DNA encoding a cell cycle protein in accordancewith established techniques and the genomic sequences used to generatetransgenic animals that contain cells which express the desired DNA.Methods for generating transgenic animals, particularly animals such asmice or rats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,particular cells would be targeted for the cell cycle protein transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding a cell cycle protein introducedinto the germ line of the animal at an embryonic stage can be used toexamine the effect of increased expression of the desired nucleic acid.Such animals can be used as tester animals for reagents thought toconfer protection from, for example, pathological conditions associatedwith its overexpression. In accordance with this facet of the invention,an animal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

[0097] Alternatively, non-human homologues of the cell cycle protein canbe used to construct a cell cycle protein “knock out” animal which has adefective or altered gene encoding a cell cycle protein as a result ofhomologous recombination between the endogenous gene encoding a cellcycle protein and altered genomic DNA encoding a cell cycle proteinintroduced into an embryonic cell of the animal. For example, cDNAencoding a cell cycle protein can be used to clone genomic DNA encodinga cell cycle protein in accordance with established techniques. Aportion of the genomic DNA encoding a cell cycle protein can be deletedor replaced with another gene, such as a gene encoding a selectablemarker which can be used to monitor integration. Typically, severalkilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector [see e.g., Thomas and Capecchi, Cell 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporafion) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the cell cycle protein.

[0098] It is understood that the models described herein can be varied.For example, “knock-in” models can be formed, or the models can becell-based rather than animal models.

[0099] Nucleic acid encoding the cell cycle polypeptides, antagonists oragonists may also be used in gene therapy. In gene therapy applications,genes are introduced into cells in order to achieve in vivo synthesis ofa therapeutically effective genetic product, for example for replacementof a defective gene. “Gene therapy” includes both conventional genetherapy where a lasting effect is achieved by a single treatment, andthe administration of gene therapeutic agents, which involves the onetime or repeated administration of a therapeutically effective DNA ormRNA. Antisense RNAs and DNAs can be used as therapeutic agents forblocking the expression of certain genes in vivo. It has already beenshown that short antisense oligonucleotides can be imported into cellswhere they act as inhibitors, despite their low intracellularconcentrations caused by their restricted uptake by the cell membrane.(Zamecnik et al., Proc. Natl. Acad. Sci. USA 83, 4143-4146 [1986]). Theoligonucleotides can be modified to enhance their uptake, e.g. bysubstituting their negatively charged phosphodiester groups by unchargedgroups.

[0100] There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro, orin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjecfion, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Thecurrently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al, Trends inBiotechnology 11 205-210 [1993]). In some situations it is desirable toprovide the nucleic acid source with an agent that targets the targetcells, such as an antibody specific for a cell surface membrane proteinor the target cell, a ligand for a receptor on the target cell, etc.Where liposomes are employed, proteins which bind to a cell surfacemembrane protein associated with endocytosis may be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

[0101] In a preferred embodiment, the cell cycle proteins, nucleicacids, variants, modified proteins, cells and/or transgenics containingthe said nucleic acids or proteins are used in screening assays.Identification of the cell cycle protein provided herein permits thedesign of drug screening assays for compounds that bind or interferewith the binding to the cell cycle protein and for compounds whichmodulate cell cycle activity.

[0102] The assays described herein preferably utilize the human cellcycle protein, although other mammalian proteins may also be used,including rodents (mice, rats, hamsters, guinea pigs, etc.), farmanimals (cows, sheep, pigs, horses, etc.) and primates. These lalterembodiments may be preferred in the development of animal models ofhuman disease. In some embodiments, as outlined herein, variant orderivative cell cycle proteins may be used, including deletion cellcycle proteins as outlined above.

[0103] In a preferred embodiment, the methods comprise combining a cellcyle protein and a candidate bioactive agent, and determining thebinding of the candidate agent to the cell cycle protein. In otherembodiments, further discussed below, binding interference orbioactivity is determined. The term “candidate bioactive agent” or“exogeneous compound” as used herein describes any molecule, e.g.,protein, small organic molecule, carbohydrates (includingpolysaccharides), polynucleotide, lipids, etc. Generally a plurality ofassay mixtures are run in parallel with different agent concentrationsto obtain a differential response to the various concentrations.Typically, one of these concentrations serves as a negative control,i.e., at zero concentration or below the level of detection. Inaddition, positive controls, i.e. the use of agents known to alter cellcycling, may be

[0104] used. For example, p21 is a molecule known to arrest cells in theG1 cell phase, by binding G1 cyclin-CDK complexes.

[0105] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 100 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pydmidines, derivatives, structural analogs orcombinations thereof. Particularly preferred are peptides.

[0106] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means. Knownpharmacological agents may be subjected to directed or random chemicalmodifications, such as acylation, alkylation, esterification,amidification to produce structural analogs.

[0107] In a preferred embodiment, a library of different candidatebioactive agents are used. Preferably, the library should provide asufficiently structurally diverse population of randomized agents toeffect a probabilistically sufficient range of diversity to allowbinding to a particular target. Accordingly, an interaction libraryshould be large enough so that at least one of its members will have astructure that gives it affinity for the target. Although it isdifficult to gauge the required absolute size of an interaction library,nature provides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷to 10⁸is sufficient to find structures with affinity for thetarget. A library of all combinations of a peptide 7 to 20 amino acidsin length, such as generally proposed herein, has the potential to codefor 20⁷ (10⁹) to 20²⁰ Thus, with libraries of 10⁷ to 10⁸ differentmolecules the present methods allow a “working” subset of atheoretically complete interaction library for 7 amino acids, and asubset of shapes for the 20²⁰ library. Thus, in a preferred embodiment,at least 10⁶, preferably at least 10⁷, more preferably at least 10⁸ andmost preferably at least 10⁹ different sequences are simultaneouslyanalyzed in the subject methods. Preferred methods maximize library sizeand diversity.

[0108] In a preferred embodiment, the candidate bioactive agents areproteins. By “protein” herein is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations. Chemical blocking groups orother chemical substituents may also be added.

[0109] In a preferred embodiment, the candidate bioactive agents arenaturally occurring proteins or fragments of naturally occurringproteins. Thus, for example, cellular extracts containing proteins, orrandom or directed digests of proteinaceous cellular extracts, may beused. In this way libraries of procaryotic and eukaryotic proteins maybe made for screening in the systems described herein. Particularlypreferred in this embodiment are libraries of bacterial, fungal, viral,and mammalian proteins, with the lafter being preferred, and humanproteins being especially preferred.

[0110] In a preferred embodiment, the candidate bioactive agents arepeptides of from about 5 to about 30 amino acids, with from about 5 toabout 20 amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

[0111] In one embodiment, the library is fully randomized, with nosequence preferences or constants at any position. In a preferredembodiment, the library is biased. That is, some positions within thesequence are either held constant, or are selected from a limited numberof possibilities. For example, in a preferred embodiment, thenucleotides or amino acid residues are randomized within a definedclass, for example, of hydrophobic amino acids, hydrophilic residues,sterically biased (either small or large) residues, towards the creationof cysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

[0112] In a preferred embodiment, the candidate bioactive agents arenucleic acids. By “nucleic acid” or “oligonucleotide” or grammaticalequivalents herein means at least two nucleotides covalently linkedtogether. A nucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage, et a., Tetrahedron, 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970);Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al.,Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805(1984), Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); andPauwels, et al., Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag,et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu, et al., J. Am. Chem. Soc.,111:2321 (1989)), O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc., 114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl.,31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et al.,Nature, 380:207 (1996), all of which are incorporated by reference)).Other analog nucleic acids include those with positive backbones(Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionicbackbones (U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141;and 4,469,863; Kiedrowshi, et al., Angew. Chem. Intl. Ed. English,30:423 (1991); Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988);Letsinger, et al., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2and 3, ASC Symposium Series 580, “Carbohydrate Modifications inAnfisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, etal., Bioorganic & Medicinal Chem. Left., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Left., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp.169-176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments. In addition, mixtures of naturally occurring nucleic acidsand analogs can be made. Alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. The nucleic acids may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo-and ribo-nucleotides, and any combination of bases,including uracil, adenine, thymine, cytosine, guanine, inosine,xathanine hypoxathanine, isocytosine, isoguanine, etc.

[0113] As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eukaryofic genomes may be used as is outlined above forproteins.

[0114] In a preferred embodiment, the candidate bioactive agents areorganic chemical moieties, a wide variety of which are available in theliterature.

[0115] In a preferred embodiment, the candidate bioactive agents arelinked to a fusion partner. By “fusion partner” or “functional group”herein is meant a sequence that is associated with the candidatebioactive agent, that confers upon all members of the library in thatclass a common function or ability. Fusion partners can be heterologous(i.e. not native to the host cell), or synthetic (not native to anycell). Suitable fusion partners include, but are not limited to: a)presentation structures, which provide the candidate bioactive agents ina conformationally restricted or stable form; b) targeting sequences,which allow the localization of the candidate bioactive agent into asubcellular or extracellular compartment; c) rescue sequences whichallow the purification or isolation of either the candidate bioactiveagents or the nucleic acids encoding them; d) stability sequences, whichconfer stability or protection from degradation to the candidatebioactive agent or the nucleic acid encoding it, for example resistanceto proteolytic degradation; e) dimerization sequences, to allow forpeptide dimerization; or f) any combination of a), b), c), d), and e),as well as linker sequences as needed.

[0116] In one embodiment of the methods described herein, portions ofcell cycle proteins are utilized; in a preferred embodiment, portionshaving cell cycle activity are used. Cell cycle activity is describedfurther below and includes binding activity to RIP3 or cell cycleprotein modulators as further described below. In addition, the assaysdescribed herein may utilize either isolated cell cycle proteins orcells comprising the cell cycle proteins.

[0117] Generally, in a preferred embodiment of the methods herein, forexample for binding assays, the cell cycle protein or the candidateagent is non-diffusibly bound to an insoluble support having isolatedsample receiving areas (e.g. a microtiter plate, an array, etc.). Theinsoluble supports may be made of any composition to which thecompositions can be bound, is readily separated from soluble material,and is otherwise compatible with the overall method of screening. Thesurface of such supports may be solid or porous and of any convenientshape. Examples of suitable insoluble supports include microtiterplates, arrays, membranes and beads. These are typically made of glass,plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose,teflon™, etc. Microtiter plates and arrays are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples. In some cases magneticbeads and the like are included. The particular manner of binding of thecomposition is not crucial so long as it is compatible with the reagentsand overall methods of the invention, maintains the activity of thecomposition and is nondiffusable. Preferred methods of binding includethe use of antibodies (which do not sterically block either the ligandbinding site or activation sequence when the protein is bound to thesupport), direct binding to “sticky” or ionic supports, chemicalcrosslinking, the synthesis of the protein or agent on the surface, etc.In some embodiments, RIP3 can be used. Following binding of the proteinor agent, excess unbound material is removed by washing. The samplereceiving areas may then be blocked through incubation with bovine serumalbumin (BSA), casein or other innocuous protein or other moiety. Alsoincluded in this invention are screening assays wherein solid supportsare not used; examples of such are described below.

[0118] In a preferred embodiment, the cell cycle protein is bound to thesupport, and a candidate bioactive agent is added to the assay.Alternatively, the candidate agent is bound to the support and the cellcycle protein is added. Novel binding agents include specificantibodies, non-natural binding agents identified in screens of chemicallibraries, peptide analogs, etc. Of particular interest are screeningassays for agents that have a low toxicity for human cells. A widevariety of assays may be used for this purpose, including labeled invitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays for protein binding, functional assays(phosphorylation assays, etc.) and the like.

[0119] The determination of the binding of the candidate bioactive agentto the cell cycle protein may be done in a number of ways. In apreferred embodiment, the candidate bioactive agent is labelled, andbinding determined directly. For example, this may be done by attachingall or a portion of the cell cycle protein to a solid support, adding alabelled candidate agent (for example a fluorescent label), washing offexcess reagent, and determining whether the label is present on thesolid support. Various blocking and washing steps may be utilized as isknown in the art.

[0120] By “labeled” herein is meant that the compound is either directlyor indirectly labeled with a label which provides a detectable signal,e.g. radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

[0121] In some embodiments, only one of the components is labeled. Forexample, the proteins (or proteinaceous candidate agents) may be labeledat tyrosine positions using ¹²⁵I, or with fluorophores. Alternatively,more than one component may be labeled with different labels; using ¹²⁵Ifor the proteins, for example, and a fluorophor for the candidateagents.

[0122] In a preferred embodiment, the binding of the candidate bioactiveagent is determined through the use of competitive binding assays. Inthis embodiment, the competitor is a binding moiety known to bind to thetarget molecule (i.e. cell cycle protein), such as an antibody, peptide,binding partner, ligand, etc. In a preferred embodiment, the competitoris RIP3. Under certain circumstances, there may be competitive bindingas between the bioactive agent and the binding moiety, with the bindingmoiety displacing the bioactive agent. This assay can be used todetermine candidate agents which interfere with binding between cellcycle proteins and RIP3. “Interference of binding” as used herein meansthat native binding of the cell cycle protein differs in the presence ofthe candidate agent. The binding can be eliminated or can be with areduced affinity. Therefore, in one embodiment, interference is causedby, for example, a conformation change, rather than direct competitionfor the native binding site.

[0123] In one embodiment, the candidate bioactive agent is labeled.Either the candidate bioactive agent, or the competitor, or both, isadded first to the protein for a time sufficient to allow binding, ifpresent. Incubations may be performed at any temperature whichfacilitates optimal activity, typically between 4 and 40° C. Incubationperiods are selected for optimum activity, but may also be optimized tofacilitate rapid high through put screening. Typically between 0.1 and 1hour will be sufficient. Excess reagent is generally removed or washedaway. The second component is then added, and the presence or absence ofthe labeled component is followed, to indicate binding.

[0124] In a preferred embodiment, the competitor is added first,followed by the candidate bioactive agent. Displacement of thecompetitor is an indication that the candidate bioactive agent isbinding to the cell cycle protein and thus is capable of binding to, andpotentially modulating, the activity of the cell cycle protein. In thisembodiment, either component can be labeled. Thus, for example, if thecompetitor is labeled, the presence of label in the wash solutionindicates displacement by the agent. Alternatively, if the candidatebioactive agent is labeled, the presence of the label on the supportindicates displacement.

[0125] In an alternative embodiment, the candidate bioactive agent isadded first, with incubation and washing, followed by the competitor.The absence of binding by the competitor may indicate that the bioactiveagent is bound to the cell cycle protein with a higher affinity. Thus,if the candidate bioactive agent is labeled, the presence of the labelon the support, coupled with a lack of competitor binding, may indicatethat the candidate agent is capable of binding to the cell cycleprotein.

[0126] In a preferred embodiment, the methods comprise differentialscreening to identity bioactive agents that are capable of modulatingthe activity of the cell cycle proteins. Such assays can be done withthe cell cycle protein or cells comprising said cell cycle protein. Inone embodiment, the methods comprise combining an cell cycle protein anda competitor in a first sample. A second sample comprises a candidatebioactive agent, an cell cycle protein and a competitor. The binding ofthe competitor is determined for both samples, and a change, ordifference in binding between the two samples indicates the presence ofan agent capable of binding to the cell cycle protein and potentiallymodulating its activity. That is, if the binding of the competitor isdifferent in the second sample relative to the first sample, the agentis capable of binding to the cell cycle protein.

[0127] Alternatively, a preferred embodiment utilizes differentialscreening to identify drug candidates that bind to the native cell cycleprotein, but cannot bind to modified cell cycle proteins. The structureof the cell cycle protein may be modeled, and used in rational drugdesign to synthesize agents that interact with that site. Drugcandidates that affect cell cycle bioactivity are also identified byscreening drugs for the ability to either enhance or reduce the activityof the protein.

[0128] Positive controls and negative controls may be used in theassays. Preferably all control and test samples are performed in atleast triplicate to obtain statistically significant results. Incubationof all samples is for a time sufficient for the binding of the agent tothe protein. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

[0129] A variety of other reagents may be included in the screeningassays. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc which may be used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Also reagents that otherwise improve the efficiency of theassay, such as protease inhibitors, nuclease inhibitors, anti-microbialagents, etc., may be used. The mixture of components may be added in anyorder that provides for the requisite binding.

[0130] Screening for agents that modulate the activity of cell cycle mayalso be done. In a preferred embodiment, methods for screening for abioactive agent capable of modulating the activity of cell cyclecomprise the steps of adding a candidate bioactive agent to a sample ofa cell cycle protein (or cells comprising a cell cycle protein) anddetermining an alteration in the biological activity of the cell cycleprotein. “Modulating the activity of a cell cycle protein” includes anincrease in activity, a decrease in activity, or a change in the type orkind of activity present. Thus, in this embodiment, the candidate agentshould both bind to cell cycle (although this may not be necessary), andalter its biological or biochemical activity as defined herein. Themethods include both in vitro screening methods, as are generallyoutlined above, and in vivo screening of cells for alterations in thepresence, distribution, activity or amount of cell cycle protein.

[0131] Thus, in this embodiment, the methods comprise combining an cellcycle sample and a candidate bioactive agent, and evaluating the effecton the cell cycle as described above or by cell cycle protein activity.By “cell cycle protein activity” or grammatical equivalents herein ismeant at least one of the cell cycle protein's biological activities,including, but not limited to, its ability to affect the cell cycle,bind to RIP3, compete with other molecules for binding to RIP3, modulateresponses to TNF, modulate NF-K B activation, modulate apoptosis,phosphorylation activity and modulate inflammation disease.

[0132] In a preferred embodiment, the activity of the cell cycle proteinis decreased; in another preferred embodiment, the activity of the cellcycle protein is increased. Thus, bioactive agents that are antagonistsare preferred in some embodiments, and bioactive agents that areagonists may be preferred in other embodiments.

[0133] In a preferred embodiment, the invention provides methods forscreening for bioactive agents capable of modulating the activity of ancell cycle protein. The methods comprise adding a candidate bioactiveagent, as defined above, to a cell comprising cell cycle proteins.Preferred cell types include almost any cell. The cells contain arecombinant nucleic acid that encodes an cell cycle protein. In apreferred embodiment, a library of candidate agents are tested on aplurality of cells.

[0134] Detection of cell cycle regulation may be done as will beappreciated by those in the art. In one embodiment, indicators of thecell cycle are used. There are a number of parameters that may beevaluated or assayed to allow the detection of alterations in cell cycleregulation, including, but not limited to, cell viability assays, assaysto determine whether cells are arrested at a particular cell cycle stage(“cell proliferation assays”), and assays to determine at which cellstage the cells have arrested (“cell phase assays”). By assaying ormeasuring one or more of these parameters, it is possible to detect notonly alterations in cell cycle regulation, but alterations of differentsteps of the cell cycle regulation pathway. This may be done to evaluatenative cells, for example to quantify the aggressiveness of a tumor celltype, or to evaluate the effect of candidate drug agents that are beingtested for their effect on cell cycle regulation. In this manner, rapid,accurate screening of candidate agents may be performed to identifyagents that modulate cell cycle regulation.

[0135] Thus, the present compositions and methods are useful toelucidate bioactive agents that can cause a cell or a population ofcells to either move out of one growth phase and into another, or arrestin a growth phase. In some embodiments, the cells are arrested in aparticular growth phase, and it is desirable to either get them out ofthat phase or into a new phase. Alternatively, it may be desirable toforce a cell to arrest in a phase, for example G1 , rather than continueto move through the cell cycle. Similarly, it may be desirable in somecircumstances to accelerate a non-arrested but slowly moving populationof cells into either the next phase or just through the cell cycle, orto delay the onset of the next phase. For example, it may be possible toalter the activities of certain enzymes, for example kinases,phosphatases, proteases or ubiquitination enzymes, that contribute toinitiating cell phase changes.

[0136] In a preferred embodiment, the methods outlined herein are doneon cells that are not arrested in the G1 phase; that is, they arerapidly or uncontrollably growing and replicating, such as tumor cells.In this manner, candidate agents are evaluated to find agents that canalter the cell cycle regulation, i.e. cause the cells to arrest at cellcycle checkpoints, such as in G1 (although arresting in other phasessuch as S, G2 or M are also desirable). Alternatively, candidate agentsare evaluated to find agents that can cause proliferation of apopulation of cells, i.e. that allow cells that are generally arrestedin G1 to start proliferating again; for example, peripheral blood cells,terminally differentiated cells, stem cells in culture, etc.

[0137] Accordingly, the invention provides methods for screening foralterations in cell cycle regulation of a population of cells. By“alteration” or “modulation” (used herein interchangeably), is generallymeant one of two things. In a preferred embodiment, the alterationresults in a change in the cell cycle of a cell, i.e. a proliferatingcell arrests in any one of the phases, or an arrested cell moves out ofits arrested phase and starts the cell cycle, as compared to anothercell or in the same cell under different conditions. Alternatively, theprogress of a cell through any particular phase may be altered; that is,there may be an acceleration or delay in the length of time it takes forthe cells to move thorough a particular growth phase. For example, thecell may be normally undergo a G1 phase of several hours; the additionof an agent may prolong the G1 phase.

[0138] The measurements can be determined wherein all of the conditionsare the same for each measurement, or under various condibons, with orwithout bioactive agents, or at different stages of the cell cycleprocess. For example, a measurement of cell cycle regulation can bedetermined in a cell or cell population wherein a candidate bioactiveagent is present and wherein the candidate bioactive agent is absent. Inanother example, the measurements of cell cycle regulation aredetermined wherein the condition or environment of the cell orpopulations of cells differ from one another. For example, the cells maybe evaluated in the presence or absence or previous or subsequentexposure of physiological signals, for example hormones, antibodies,peptides, antigens, cytokines, growth factors, action potentials,pharmacological agents including chemotherapeutics, radiation,carcinogenics, or other cells (i.e. cell-cell contacts). In anotherexample, the measurements of cell cycle regulation are determined atdifferent stages of the cell cycle process. In yet another example, themeasurements of cell cycle regulation are taken wherein the conditionsare the same, and the alterations are between one cell or cellpopulation and another cell or cell population.

[0139] By a “population of cells” or “library of cells” herein is meantat least two cells, with at least about 10³ being preferred, at leastabout 10⁶ being particularly preferred, and at least about 10⁸ to 10⁹being especially preferred. The population or sample can contain amixture of different cell types from either primary or secondarycultures although samples containing only a single cell type arepreferred, for example, the sample can be from a cell line, particularlytumor cell lines, as outlined below. The cells may be in any cell phase,either synchronously or not, including M, G1 , S, and G2. In a preferredembodiment, cells that are replicating or proliferating are used; thismay allow the use of retroviral vectors for the introduction ofcandidate bioactve agents. Alternauvely, non-replicating cells may beused, and other vectors (such as adenovirus and lentivirus vectors) canbe used. In addition, although not required, the cells are compatiblewith dyes and antibodies.

[0140] Preferred cell types for use in the invention include, but arenot limited to, mammalian cells, including animal (rodents, includingmice, rats, hamsters and gerbils), primates, and human cells,particularly including tumor cells of all types, including breast, skin,lung, cervix, colonrectal, leukemia, brain, etc.

[0141] In a preferred embodiment, the methods comprise assaying one ormore of several different cell parameters, including, but not limitedto, cell viability, cell proliferation, and cell phase.

[0142] In a preferred embodiment, cell viability is assayed, to ensurethat a lack of cellular change is due to experimental conditions (i.e.the introduction of a candidate bioactive agent) not cell death. Thereare a variety of suitable cell viability assays which can be used,including, but not limited to, light scattering, viability dye staining,and exclusion dye staining.

[0143] In a preferred embodiment, a light scattering assay is used asthe viability assay, as is well known in the art. For example, whenviewed in the FACS, cells have particular characteristics as measured bytheir forward and 90 degree (side) light scatter properties. Thesescatter properties represent the size, shape and granule content of thecells. These properties account for two parameters to be measured as areadout for the viability. Briefly, the DNA of dying or dead cellsgenerally condenses, which alters the 90° scatter; similarly, membraneblebbing can alter the forward scatter. Alterations in the intensity oflight scattering, or the cell-refractive index indicate alterations inviability.

[0144] Thus, in general, for light scattering assays, a live cellpopulation of a particular cell type is evaluated to determine it'sforward and side scattering properties. This sets a standard forscattering that can subsequently be used.

[0145] In a preferred embodiment, the viability assay utilizesamiability dye. There are a number of known viability dyes that staindead or dying cells, but do not stain growing cells. For example,annexin V is a member of a protein family which displays specificbinding to phospholipid (phosphotidylserine) in a divalent ion dependentmanner. This protein has been widely used for the measurement ofapoptosis (programmed cell death) as cell surface exposure ofphosphatidylserine is a hallmark early signal of this process. Suitableviability dyes include, but are not limited to, annexin, ethidiumhomodimer-1, DEAD Red, propidium iodide, SYTOX Green, etc., and othersknown in the art; see the Molecular Probes Handbook of FluorescentProbes and Research Chemicals, Haugland, Sixth Edition, herebyincorporated by reference; see Apoptosis Assay on page 285 inparticular, and Chapter 16.

[0146] Protocols for viability dye staining for cell viability areknown, see Molecular Probes catalog, supra. In this embodiment, theviability dye such as annexin is labeled, either directly or indirectly,and combined with a cell population. Annexin is commercially available,i.e., from PharMingen, San Diego, Calif., or Caltag Laboratories,Millbrae, Calif. Preferably, the viability dye is provided in a solutionwherein the dye is in a concentration of about 100 ng/ml to about 500ng/ml, more preferably, about 500 ng/ml to about 1 μg/ml, and mostpreferably, from about 1 μg/ml to about 5 μg/ml. In a preferredembodiment, the viability dye is directly labeled; for example, annexinmay be labeled with a fluorochrome such as fluorecein isothiocyanate(FITC), Alexa dyes, TRITC, AMCA, APC, tri-color, Cy-5, and others knownin the art or commercially available. In an alternate preferredembodiment, the viability dye is labeled with a first label, such as ahapten such as biotin, and a secondary fluorescent label is used, suchas fluorescent streptavidin. Other first and second labeling pairs canbe used as will be appreciated by those in the art.

[0147] Once added, the viability dye is allowed to incubate with thecells for a period of time, and washed, if necessary. The cells are thensorted as outlined below to remove the non-viable cells.

[0148] In a preferred embodiment, exclusion dye staining is used as theviability assay. Exclusion dyes are those which are excluded from livingcells, i.e. they are not taken up passively (they do not permeate thecell membrane of a live cell). However, due to the permeability of deador dying cells, they are taken up by dead cells. Generally, but notalways, the exclusion dyes bind to DNA, for example via intercalation.Preferably, the exclusion dye does not fluoresce, or fluoresces poorly,in the absence of DNA; this eliminates the need for a wash step.Alternatively, exclusion dyes that require the use of a secondary labelmay also be used. Preferred exclusion dyes include, but are not limitedto, ethidium bromide; ethidium homodimer-1; propidium iodine; SYTOXgreen nucleic acid stain; Calcein AM, BCECF AM; fluorescein diacetate;TOTO® and TO-PRO™ (from Molecular Probes; supra, see chapter 16) andothers known in the art.

[0149] Protocols for exclusion dye staining for cell viability areknown, see the Molecular Probes catalog, supra. In general, theexclusion dye is added to the cells at a concentration of from about 100ng/ml to about 500 ng/ml, more preferably, about 500 ng/ml to about 1μg/ml, and most preferably, from about 0.1 μg/ml to about 5 μg/ml, withabout 0.5 μg/mi being particularly preferred. The cells and theexclusion dye are incubated for some period of time, washed, ifnecessary, and then the cells sorted as outlined below, to removenon-viable cells from the population.

[0150] In addition, there are other cell viability assays which may berun, including for example enzymatic assays, which can measureextracellular enzymatic activity of either live cells (i.e. secretedproteases, etc.), or dead cells (i.e. the presence of intracellularenzymes in the media; for example, intracellular proteases,mitochondrial enzymes, etc.). See the Molecular Probes Handbook ofFluorescent Probes and Research Chemicals, Haugland, Sixth Edition,hereby incorporated by reference; see chapter 16 in particular.

[0151] In a preferred embodiment, at least one cell viability assay isrun, with at least two different cell viability assays being preferred,when the fluors are compatible. When only 1 viability assay is run, apreferred embodiment utilizes light scattering assays (both forward andside scattering). When two viability assays are run, preferredembodiments utilize light scattering and dye exclusion, with lightscattering and viability dye staining also possible, and all three beingdone in some cases as well. Viability assays thus allow the separationof viable cells from non-viable or dying cells.

[0152] In addition to a cell viability assay, a preferred embodimentutilizes a cell proliferation assay. By “proliferation assay” herein ismeant an assay that allows the determination that a cell population iseither proliferating, i.e. replicating, or not replicating.

[0153] In a preferred embodiment, the proliferation assay is a dyeinclusion assay. A dye inclusion assay relies on dilution effects todistinguish between cell phases. Briefly, a dye (generally a fluorescentdye as outlined below) is introduced to cells and taken up by the cells.Once taken up, the dye is trapped in the cell, and does not diffuse out.As the cell population divides, the dye is proportionally diluted. Thatis, after the introduction of the inclusion dye, the cells are allowedto incubate for some period of time; cells that lose fluorescence overtime are dividing, and the cells that remain fluorescent are arrested ina non-growth phase.

[0154] Generally, the introduction of the inclusion dye may be done inone of two ways. Either the dye cannot passively enter the cells (e.g.it is charged), and the cells must be treated to take up the dye; forexample through the use of a electric pulse. Alternatively, the dye canpassively enter the cells, but once taken up, it is modified such thatit cannot diffuse out of the cells. For example, enzymatic modificationof the inclusion dye may render it charged, and thus unable to diffuseout of the cells. For example, the Molecular Probes CellTracker™ dyesare fluorescent chloromethyl derivatives that freely diffuse into cells,and then glutathione S-transferase-mediated reaction produces membraneimpermeant dyes.

[0155] Suitable inclusion dyes include, but are not limited to, theMolecular Probes line of CellTracker™ dyes , including, but not limitedto CellTracker™ Blue, CellTracker™ Yellow-Green, CellTracker™ Green,CellTracker™ Orange, PKH26 (Sigma), and others known in the art; see theMolecular Probes Handbook, supra; chapter 15 in particular.

[0156] In general, inclusion dyes are provided to the cells at aconcentration ranging from about 100 ng/ml to about 5 μg/ml, with fromabout 500 ng/ml to about 1 μg/ml being preferred. A wash step may or maynot be used. In a preferred embodiment, a candidate bioactive agent iscombined with the cells as described herein. The cells and the inclusiondye are incubated for some period of time, to allow cell division andthus dye dilution. The length of time will depend on the cell cycle timefor the particular cells; in general, at least about 2 cell divisionsare preferred, with at least about 3 being particularly preferred and atleast about 4 being especially preferred. The cells are then sorted asoutlined below, to create populations of cells that are replicating andthose that are not. As will be appreciated by those in the art, in somecases, for example when screening for anti-proliferation agents, thebright (i.e. fluorescent) cells are collected; in other embodiments, forexample for screening for proliferation agents, the low fluorescencecells are collected. Alterations are determined by measuring thefluorescence at either different time points or in different cellpopulations, and comparing the determinations to one another or tostandards.

[0157] In a preferred embodiment, the proliferation assay is anantimetabolite assay. In general, antimetabolite assays find the mostuse when agents that cause cellular arrest in G1 or G2 resting phase isdesired. In an antimetabolite proliferation assay, the use of a toxicantimetabolite that will kill dividing cells will result in survival ofonly those cells that are not dividing. Suitable antimetabolitesinclude, but are not limited to, standard chemotherapeutic agents suchas methotrexate, cisplatin, taxol, hydroxyurea, nucleotide analogs suchas AraC, etc. In addition, antimetabolite assays may include the use ofgenes that cause cell death upon expression.

[0158] The concentration at which the antimetabolite is added willdepend on the toxicity of the particular antimetabolite, and will bedetermined as is known in the art. The antimetabolite is added and thecells are generally incubated for some period of time; again, the exactperiod of time will depend on the characteristics and identity of theantimetabolite as well as the cell cycle time of the particular cellpopulation. Generally, a time sufficient for at least one cell divisionto occur.

[0159] In a preferred embodiment, at least one proliferation assay isrun, with more than one being preferred. Thus, a proliferation assayresults in a population of proliferating cells and a population ofarrested cells. Moreover, other proliferation assays may be used, i.e.,colorimetric assays known in the art.

[0160] In a preferred embodiment, either after or simultaneously withone or more of the proliferation assays outlined above, at least onecell phase assay is done. A “cell phase” assay determines at which cellphase the cells are arrested, M, G1 , S, or G2.

[0161] In a preferred embodiment, the cell phase assay is a DNA bindingdye assay. Briefly, a DNA binding dye is introduced to the cells, andtaken up passively. Once inside the cell, the DNA binding dye binds toDNA, generally by intercalation, although in some cases, the dyes can beeither major or minor groove binding compounds. The amount of dye isthus directly correlated to the amount of DNA in the cell, which variesby cell phase; G2 and M phase cells have twice the DNA content of G1phase cells, and S phase cells have an intermediate amount, depending onat what point in S phase the cells are. Suitable DNA binding dyes arepermeant, and include, but are not limited to, Hoechst 33342 and 33258,acridine orange, 7-AAD, LDS 751, DAPI, and SYTO 16, Molecular ProbesHandbook, supra; chapters 8 and 16 in particular.

[0162] In general, the DNA binding dyes are added in concentrationsranging from about 1

[0163] μg/ml to about 5 μg/ml. The dyes are added to the cells andallowed to incubate for some period of time; the length of time willdepend in part on the dye chosen. In one embodiment, measurements aretaken immediately after addition of the dye. The cells are then sortedas outlined below, to create populations of cells that contain differentamounts of dye, and thus different amounts of DNA; in this way, cellsthat are replicating are separated from those that are not. As will beappreciated by those in the art, in some cases, for example whenscreening for anti-proliferation agents, cells with the leastfluorescence (and thus a single copy of the genome) can be separatedfrom those that are replicating and thus contain more than a singlegenome of DNA. Alterations are determined by measuring the fluorescenceat either different time points or in different cell populations, andcomparing the determinations to one another or to standards.

[0164] In a preferred embodiment, the cell phase assay is a cyclindestruction assay. In this embodiment, prior to screening (and generallyprior to the introduction of a candidate bioactive agent, as outlinedbelow), a fusion nucleic acid is introduced to the cells. The fusionnucleic acid comprises nucleic acid encoding a cyclin destruction boxand a nucleic acid encoding a detectable molecule. “Cyclin destructionboxes” are known in the art and are sequences that cause destruction viathe ubiquitination pathway of proteins containing the boxes duringparticular cell phases. That is, for example, G1 cyclins may be stableduring G1 phase but degraded during S phase due to the presence of a G1cyclin destruction box. Thus, by linking a cyclin destruction box to adetectable molecule, for example green fluorescent protein, the presenceor absence of the detectable molecule can serve to identify the cellphase of the cell population. In a preferred embodiment, multiple boxesare used, preferably each with a different fluor, such that detection ofthe cell phase can occur.

[0165] A number of cyclin destruction boxes are known in the art, forexample, cyclin A has a destruction box comprising the sequenceRTVLGVIGD; the destruction box of cyclin B1 comprises the sequenceRTALGDIGN. See Glotzer et al., Nature 349:132-138 (1991). Otherdestruction boxes are known as well: YMTVSIIDRFMQDSCVPKKMLQLVGVT (ratcyclin B); KFRLLQETMYMTVSIIDRFMQNSCVPKK (mouse cyclin B);RAILIDWLIQVQMKFRLLQETMYMTVS (mouse cyclin B 1);DRFLQAQLVCRKKLQWGITALLLASK (mouse cyclin B2); and MSVLRGKLQLVGTMMLL(mouse cyclin A2).

[0166] The nucleic acid encoding the cyclin destruction box is operablylinked to nucleic acid encoding a detectable molecule. The fusionproteins are constructed by methods known in the art. For example, thenucleic acids encoding the destruction box is ligated to a nucleic acidencoding a detectable molecule. By “detectable molecule” herein is meanta molecule that allows a cell or compound comprising the detectablemolecule to be distinguished from one that does not contain it, i.e., anepitope, sometimes called an antigen TAG, a specific enzyme, or afluorescent molecule. Preferred fluorescent molecules include but arenot limited to green fluorescent protein (GFP), blue fluorescent protein(BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP),and enzymes including luciferase and P-galactosidase. When antigen TAGsare used, preferred embodiments utilize cell surface antigens. Theepitope is preferably any detectable peptide which is not generallyfound on the cytoplasmic membrane, although in some instances, if theepitope is one normally found on the cells, increases may be detected,although this is generally not preferred. Similarly, enzymaticdetectable molecules may also be used; for example, an enzyme thatgenerates a novel or chromogenic product.

[0167] Accordingly, the results of sorting after cell phase assaysgenerally result in at least two populations of cells that are indifferent cell phases.

[0168] The proteins and nucleic acids provided herein can also be usedfor screening purposes wherein the protein-protein interactions of thecell cycle proteins can be identified. Genetic systems have beendescribed to detect protein-protein interactions. The first work wasdone in yeast systems, namely the “yeast two-hybrid” system. The basicsystem requires a protein-protein interaction in order to turn ontranscription of a reporter gene. Subsequent work was done in mammaliancells. See Fields et al., Nature 340:245 (1989); Vasavada et al., PNASUSA 88:10686 (1991); Fearon et al., PNAS USA 89:7958 (1992); Dang etal., Mol. Cell. Biol. 11:954 (1991); Chien et al., PNAS USA 88:9578(1991); and U.S. Pat. Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490,and 5,637,463. a preferred system is described in Ser. Nos. 09/050,863,filed Mar. 30, 1998 and 09/359,081 filed Jul. 22, 1999, entitled“Mammalian Protein Interaction Cloning System”. For use in conjunctionwith these systems, a particularly useful shuttle vector is described inSer. No. 09/133,944, filed Aug. 14, 1998, entitled “Shuitle Vectors”.

[0169] In general, two nucleic acids are transformed into a cell, whereone is a “bait” such as the gene encoding a cell cycle protein or aportion thereof, and the other encodes a test candidate. Only if the twoexpression products bind to one another will an indicator, such as afluorescent protein, be expressed. Expression of the indicator indicateswhen a test candidate binds to the cell cycle protein and can beidentified as an cell cycle protein. Using the same system and theidentified cell cycle proteins the reverse can be performed. Namely, thecell cycle proteins provided herein can be used to identify new baits,or agents which interact with cell cycle proteins. Additionally, thetwo-hybrid system can be used wherein a test candidate is added inaddition to the bait and the cell cycle protein encoding nucleic acidsto determine agents which interfere with the bait, such as RIP3, and thecell cycle protein.

[0170] In one embodiment, a mammalian two-hybrid system is preferred.Mammalian systems provide post-translational modifications of proteinswhich may contribute significantly to their ability to interact. Inaddition, a mammalian two-hybrid system can be used in a wide variety ofmammalian cell types to mimic the regulation, induction, processing,etc. of specific proteins within a particular cell type. For example,proteins involved in a disease state (i.e., cancer, apoptosis relateddisorders) could be tested in the relevant disease cells. Similarly, fortesting of random proteins, assaying them under the relevant cellularconditions will give the highest positive results. Furthermore, themammalian cells can be tested under a variety of experimental conditionsthat may affect intracellular protein-protein interactions, such as inthe presence of hormones, drugs, growth factors and cytokines,radiation, chemotherapeutics, cellular and chemical stimuli, etc., thatmay contribute to conditions which can effect protein-proteininteractions, particularly those involved in cancer.

[0171] Assays involving binding such as the two-hybrid system may takeinto account non-specific binding proteins (NSB).

[0172] Expression in various cell types, and assays for cell cycleactivity are described above. The activity assays, such as binding toRIP3 in situ, modulation of RIP effects, modulation of TNF response,modulation of NF-K B activation, and modulation of apoptosis can beperformed to confirm the activity of cell cycle proteins which havealready been identified by their sequence identity/similarity or bindingto RIP3, as well as to further confirm the activity of lead compoundsidentified as modulators of cell cycle protein activity.

[0173] The components provided herein for the assays provided herein mayalso be combined to form kits. The kits can be based on the use of theprotein and/or the nucleic acid encoding the cell cycle proteins. In oneembodiment, other components are provided in the kit. Such componentsinclude one or more of packaging, instructions, antibodies, and labels.Additional assays such as those used in diagnostics are furtherdescribed below.

[0174] In this way, bioactive agents are identified. Compounds withpharmacological activity are able to enhance or interfere with theactivity of the cell cycle protein. The compounds having the desiredpharmacological activity may be administered in a physiologicallyacceptable carrier to a host, as further described below.

[0175] The present discovery relating to the role of cell cycle proteinsin the cell cycle thus provides methods for inducing or preventing cellproliferation in cells. In a preferred embodiment, the cell cycleproteins, and particularly cell cycle protein fragments, are useful inthe study or treatment of conditions which are mediated by the cellcycle proteins, i.e. to diagnose, treat or prevent cell cycle associateddisorders. Thus, “cell cycle associated disorders” or “disease state”include conditions involving both insufficient or excessive cellproliferation. Examples include cancer, and inflammation disorders.

[0176] Thus, in one embodiment, cell cycle regulation in cells ororganisms are provided. In one embodiment, the methods compriseadministering to a cell or individual in need thereof, a cell cycleprotein in a therapeutic amount. Alternatively, an anti-cell cycleantibody that reduces or eliminates the biological activity of theendogeneous cell cycle protein is administered. In another embodiment, abioactive agent as identified by the methods provided herein isadministered. Alternatively, the methods comprise administering to acell or individual a recombinant nucleic acid encoding an cell cycleprotein. As will be appreciated by those in the art, this may beaccomplished in any number of ways. In a preferred embodiment, theactivity of cell cycle is increased by increasing the amount of cellcycle in the cell, for example by overexpressing the endogeneous cellcycle or by administering a gene encoding a cell cycle protein, usingknown gene-therapy techniques, for example. In a preferred embodiment,the gene therapy techniques include the incorporation of the exogeneousgene using enhanced homologous recombination (EHR), for example asdescribed in PCT/US93/03868, hereby incorporated by reference in itsentirety.

[0177] Without being bound by theory, it appears that cell cycle proteinis an important protein in the cell cycle. Accordingly, disorders basedon mutant or variant cell cycle genes may be determined. In oneembodiment, the invention provides methods for identifying cellscontaining variant cell cycle genes comprising determining all or partof the sequence of at least one endogeneous cell cycle genes in a cell.As will be appreciated by those in the art, this may be done using anynumber of sequencing techniques. In a preferred embodiment, theinvention provides methods of identifying the cell cycle genotype of anindividual comprising determining all or part of the sequence of atleast one cell cycle gene of the individual. This is generally done inat least one tissue of the individual, and may include the evaluation ofa number of tissues or different samples of the same tissue. The methodmay include comparing the sequence of the sequenced cell cycle gene to aknown cell cycle gene, i.e. a wild-type gene.

[0178] The sequence of all or part of the cell cycle gene can then becompared to the sequence of a known cell cycle gene to determine if anydifferences exist. This can be done using any number of known sequenceidentity programs, such as Bestfit, etc. In a preferred embodiment, thepresence of a difference in the sequence between the cell cycle gene ofthe patient and the known cell cycle gene is indicative of a diseasestate or a propensity for a disease state.

[0179] In one embodiment, the invention provides methods for diagnosinga cell cycle related condition in an individual. The methods comprisemeasuring the activity of cell cycle in a tissue from the individual orpatient, which may include a measurement of the amount or specificactivity of a cell cycle protein. This activity is compared to theactivity of cell cycle from either a unaffected second individual orfrom an unaffected tissue from the first individual. When theseactivities are different, the first individual may be at risk for a cellcycle associated disorder. In this way, for example, monitoring ofvarious disease conditions may be done, by monitoring the levels of theprotein or the expression of mRNA therefor. Similarly, expression levelsmay correlate to the prognosis.

[0180] In one aspect, the expression levels of cell cycle protein genesare determined in different patient samples or cells for which eitherdiagnosis or prognosis information is desired. Gene expressionmonitoring is done on genes encoding cell cycle proteins. In one aspect,the expression levels of cell cycle protein genes are determined fordifferent cellular states, such as normal cells and cells undergoingapoptosis or transformation. By comparing cell cycle protein geneexpression levels in cells in different states, information includingboth up-and down-regulation of cell cycle protein genes is obtained,which can be used in a number of ways. For example, the evaluation of aparticular treatment regime may be evaluated: does a chemotherapeuticdrug act to improve the long-term prognosis in a particular patient.Similarly, diagnosis may be done or confirmed by comparing patientsamples. Furthermore, these gene expression levels allow screening ofdrug candidates with an eye to mimicking or altering a particularexpression level. This may be done by making biochips comprising sets ofimportant cell cycle protein genes, such as those of the presentinvention, which can then be used in these screens. These methods canalso be done on the protein basis; that is, protein expression levels ofthe cell cycle proteins can be evaluated for diagnostic purposes or toscreen candidate agents. In addition, the cell cycle protein nucleicacid sequences can be administered for gene therapy purposes, includingthe administration of antisense nucleic acids, or the cell cycleproteins administered as therapeutic drugs.

[0181] Cell cycle protein sequences bound to biochips include bothnucleic acid and amino acid sequences as defined above. In a preferredembodiment, nucleic acid probes to cell cycle protein nucleic acids(both the nucleic acid sequences having the sequences outlined in theFigures and/or the complements thereof) are made. The nucleic acidprobes attached to the biochip are designed to be substantiallycomplementary to the cell cycle protein nucleic acids, i.e. the targetsequence (either the target sequence of the sample or to other probesequences, for example in sandwich assays), such that hybridization ofthe target sequence and the probes of the present invention occurs. Asoutlined below, this complementarity need not be perfect; there may beany number of base pair mismatches which will interfere withhybridization between the target sequence and the single strandednucleic acids of the present invention. However, if the number ofmutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, by “substantially complementary”0herein is meant that the probes are sufficiently complementary to thetarget sequences to hybridize under normal reaction conditions,particularly high stringency conditions, as outlined herein.

[0182] A “nucleic acid probe” is generally single stranded but can bepartially single and partially double stranded. The strandedness of theprobe is dictated by the structure, composition, and properties of thetarget sequence. In general, the nucleic acid probes range from about 8to about 100 bases long, with from about 10 to about 80 bases beingpreferred, and from about 30 to about 50 bases being particularlypreferred. In some embodiments, much longer nucleic acids can be used,up to hundreds of bases (e.g., whole genes).

[0183] As will be appreciated by those in the art, nucleic acids can beattached or immobilized to a solid support in a wide variety of ways. By“immobilized” and grammatical equivalents herein is meant theassociation or binding between the nucleic acid probe and the solidsupport is sufficient to be stable under the conditions of binding,washing, analysis, and removal as outlined below. The binding can becovalent or non-covalent. By “non-covalent binding” and grammaticalequivalents herein is meant one or more of either electrostatic,hydrophilic, and hydrophobic interactions. Included in non-covalentbinding is the covalent attachment of a molecule, such as, streptavidinto the support and the non-covalent binding of the biotinylated probe tothe streptavidin. By “covalent binding” and grammatical equivalentsherein is meant that the two moieties, the solid support and the probe,are attached by at least one bond, including sigma bonds, pi bonds andcoordination bonds. Covalent bonds can be formed directly between theprobe and the solid support or can be formed by a cross linker or byinclusion of a specific reactive group on either the solid support orthe probe or both molecules. Immobilization may also involve acombination of covalent and non-covalent interactions.

[0184] In general, the probes are attached to the biochip in a widevariety of ways, as will be appreciated by those in the art. Asdescribed herein, the nucleic acids can either be synthesized first,with subsequent attachment to the biochip, or can be directlysynthesized on the biochip.

[0185] The biochip comprises a suitable solid substrate. By “substrate”or “solid support” or other grammatical equivalents herein is meant anymaterial that can be modified to contain discrete individual sitesappropriate for the attachment or association of the nucleic acid probesand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates are very large,and include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, etc. In general, the substrates allow optical detection and donot appreciably show fluorescence.

[0186] In a preferred embodiment, the surface of the biochip and theprobe may be derivatized with chemical functional groups for subsequentattachment of the two. Thus, for example, the biochip is derivatizedwith a chemical functional group including, but not limited to, aminogroups, carboxy groups, oxo groups and thiol groups, with amino groupsbeing particularly preferred. Using these functional groups, the probescan be attached using functional groups on the probes. For example,nucleic acids containing amino groups can be attached to surfacescomprising amino groups, for example using linkers as are known in theart; for example, homo-or hetero-bifunctional linkers as are well known(see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference). Inaddition, in some cases, additional linkers, such as alkyl groups(including substituted and heteroalkyl groups) may be used.

[0187] In this embodiment, oligonucleotides, corresponding to thenucleic acid probe, are synthesized as is known in the art, and thenattached to the surface of the solid support. As will be appreciated bythose skilled in the art, either the 5′ or 3′ terminus may be attachedto the solid support, or attachment may be via an internal nucleoside.

[0188] In an additional embodiment, the immobilization to the solidsupport may be very strong, yet non-covalent. For example, biotinylatedoligonucleotides can be made, which bind to surfaces covalently coatedwith streptavidin, resulting in attachment.

[0189] Alternatively, the oligonucleotides may be synthesized on thesurface, as is known in the art. For example, photoactivation techniquesutilizing photopolymerization compounds and techniques are used. In apreferred embodiment, the nucleic acids can be synthesized in situ,using well known photolithographic techniques, such as those describedin WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; andreferences cited within, all of which are expressly incorporated byreference; these methods of attachment form the basis of the AffimetrixGeneChip™ technology.

[0190] “Differential expression,” or grammatical equivalents as usedherein, refers to both qualitative as, well as quantitative differencesin the genes' temporal and/or cellular expression patterns within andamong the cells. Thus, a differentially expressed gene can qualitativelyhave its expression altered, including an activation or inactivation,in, for example, normal versus apoptotic cell. That is, genes may beturned on or turned off in a particular state, relative to anotherstate. As is apparent to the skilled artisan, any comparison of two ormore states can be made. Such a qualitatively regulated gene willexhibit an expression pattern within a state or cell type which isdetectable by standard techniques in one such state or cell type, but isnot detectable in both. Alternatively, the determination is quantitativein that expression is increased or decreased; that is, the expression ofthe gene is either upregulated, resulting in an increased amount oftranscript, or downregulated, resulting in a decreased amount oftranscript. The degree to which expression differs need only be largeenough to quantify via standard characterization techniques as outlinedbelow, such as by use of Affymetrix GeneChip™ expression arrays,Lockhart, Nature Biotechnology 14:1675-1680 (1996), hereby expresslyincorporated by reference. Other techniques include, but are not limitedto, quantitative reverse transcriptase PCR, Northern analysis and RNaseprotection.

[0191] As will be appreciated by those in the art, this may be done byevaluation at either the gene transcript, or the protein level; that is,the amount of gene expression may be monitored using nucleic acid probesto the DNA or RNA equivalent of the gene transcript, and thequantification of gene expression levels, or, alternatively, the finalgene product itself (protein) can be monitored, for example through theuse of antibodies to the cell cycle protein and standard immunoassays(ELISAs, etc.) or other techniques, including mass spectroscopy assays,2D gel electrophoresis assays, etc.

[0192] In another method detection of the mRNA is performed in situ. Inthis method permeabilized cells or tissue samples are contacted with adetectably labeled nucleic acid probe for sufficient time to allow theprobe to hybridize with the target mRNA. Following washing to remove thenon-specifically bound probe, the label is detected. For example adigoxygenin labeled riboprobe (RNA probe) that is complementary to themRNA encoding an cell cycle protein is detected by binding thedigoxygenin with an anti-digoxygenin secondary antibody and developedwith nitro blue tetrazolium and 5-bromo4-chloro-3-indoyl phosphate.

[0193] In another preferred method, expression of cell cycle protein isperformed using in situ imaging techniques employing antibodies to cellcycle proteins. In this method cells are contacted with from one to manyantibodies to the cell cycle protein(s). Following washing to removenon-specific antibody binding, the presence of the antibody orantibodies is detected. In one embodiment the antibody is detected byincubating with a secondary antibody that contains a detectable label.In another method the primary antibody to the cell cycle protein(s)contains a detectable label. In another preferred embodiment each one ofmultiple primary antibodies contains a distinct and detectable label.This method finds particular use in simultaneous screening for aplurality of cell cycle proteins. The label may be detected in afluorometer which has the ability to detect and distinguish emissions ofdifferent wavelengths. In addition, a fluorescence activated cell sorter(FACS) can be used in this method. As will be appreciated by one ofordinary skill in the art, numerous other histological imagingtechniques are useful in the invention and the antibodies can be used inELISA, immunoblotting (Western blotting), immunoprecipitation, BIACOREtechnology, and the like.

[0194] In one embodiment, the cell cycle proteins of the presentinvention may be used to generate polyclonal and monoclonal antibodiesto cell cycle proteins, which are useful as described herein. Similarly,the cell cycle proteins can be coupled, using standard technology, toaffinity chromatography columns. These columns may then be used topurify cell cycle antibodies. In a preferred embodiment, the antibodiesare generated to epitopes unique to the cell cycle protein; that is, theantibodies show little or no cross-reactivity to other proteins. Theseantibodies find use in a number of applications. For example, the cellcycle antibodies may be coupled to standard affinity chromatographycolumns and used to purify cell cycle proteins as further describedbelow. The antibodies may also be used as blocking polypeptides, asoutlined above, since they will specifically bind to the cell cycleprotein.

[0195] The anti-cell cycle protein antibodies may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan. Polyclonal antibodies can be raised in a mammal, forexample, by one or more injections of an immunizing agent and, ifdesired, an adjuvant. Typically, the immunizing agent and/or adjuvantwill be injected in the mammal by multiple subcutaneous orintraperitoneal injections. The immunizing agent may include the cellcycle protein or a fusion protein thereof. It may be useful to conjugatethe immunizing agent to a protein known to be immunogenic in the mammalbeing immunized. Examples of such immunogenic proteins include but arenot limited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid a, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

[0196] The anti-cell cycle protein antibodies may, alternatively, bemonoclonal antibodies. Monoclonal antibodies may be prepared usinghybridoma methods, such as those described by Kohler and Milstein,Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, orother appropriate host animal, is typically immunized with an immunizingagent to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

[0197] The immunizing agent will typically include the cell cycleprotein or a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell [Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-103]. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

[0198] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, an- are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. lmmunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., N.Y., (1987) pp. 51-63].

[0199] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst cell cycle protein. Preferably, the binding specificity ofmonoclonal antibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0200] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0201] The monoclonal antibodies secreted by the subcdones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein a-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0202] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0203] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0204] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0205] The anti-cell cycle protein antibodies of the invention mayfurther comprise humanized antibodies or human antibodies. Humanizedforms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

[0206] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239.1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0207] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

[0208] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the cell cycle protein, the other one is for anyother antigen, and preferably for a cell-surface protein or receptor orreceptor subunit.

[0209] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0210] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0211] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0212] The anti-cell cycle protein antibodies of the invention havevarious utilities. For example, anti-cell cycle protein antibodies maybe used in diagnostic assays for an cell cycle protein, e.g., detectingits expression in specific cells, tissues, or serum. Various diagnosticassay techniques known in the art may be used, such as competitivebinding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: a Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or 125I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,betagalactosidase or horseradish peroxidase. Any method known in the artfor conjugating the antibody to the detectable moiety may be employed,including those methods described by Hunter et al., Nature, 144:945(1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J.Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. and Cytochem.,30:407 (1982).

[0213] Anti-Cell cycle protein antibodies also are useful for theaffinity purification of cell cycle protein from recombinant cellculture or natural sources. In this process, the antibodies against cellcycle protein are immobilized on a suitable support, such a Sephadexresin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing the cellcycle protein to be purified, and thereafter the support is washed witha suitable solvent that will remove substantially all the material inthe sample except the cell cycle protein, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the cell cycle protein from theantibody.

[0214] The anti-cell cycle protein antibodies may also be used intreatment. In one embodiment, the genes encoding the antibodies areprovided, such that the antibodies bind to and modulate the cell cycleprotein within the cell.

[0215] In one embodiment, a therapeutically effective dose of an cellcycle protein, agonist or antagonist is administered to a patient. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. As is known in the art, adjustmentsfor cell cycle protein degradation, systemic versus localized delivery,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

[0216] A “patient” for the purposes of the present invention includesboth humans and other animals, particularly mammals, and organisms. Thusthe methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

[0217] The administration of the cell cycle protein, agonist orantagonist of the present invention can be done in a variety of ways,including, but not limited to, orally, subcutaneously, intravenously,intranasally, transdermally, intraperitoneally, intramuscularly,intrapulmonary, vaginally, rectally, or intraocularly. In someinstances, for example, in the treatment of wounds and inflammation, thecomposition may be directly applied as a solution or spray. Dependingupon the manner of introduction, the compounds may be formulated in avariety of ways. The concentration of therapeutically active compound inthe formulation may vary from about 0.1-100 wt. %.

[0218] The pharmaceutical compositions of the present invention comprisean cell cycle protein, agonist or antagonist (including antibodies andbioactive agents as described herein) in a form suitable foradministration to a patient. In the preferred embodiment, thepharmaceutical compositions are in a water soluble form, such as beingpresent as pharmaceutically acceptable salts, which is meant to includeboth acid and base addition salts. “Pharmaceutically acceptable acidaddition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid andthe like, and organic acids such as acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,ptoluenesulfonic acid, salicylic acid and the like. “Pharmaceuticallyacceptable base addition salts” include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Particularlypreferred are the ammonium, potassium, sodium, calcium, and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine.

[0219] The pharmaceutical compositions may also include one or more ofthe following: carrier proteins such as serum albumin; buffers; fillerssuch as microcrystalline cellulose, lactose, corn and other starches;binding agents; sweeteners and other flavoring agents; coloring agents;and polyethylene glycol. Additives are well known in the art, and areused in a variety of formulations.

[0220] Combinations of the compositions may be administered. Moreover,the compositions may be administered in combination with othertherapeutics, including growth factors or chemotherapeutics and/orradiation. Targeting agents (i.e. ligands for receptors on cancer cells)may also be combined with the compositions provided herein.

[0221] In one embodiment provided herein, the antibodies are used forimmunotherapy, thus, methods of immunotherapy are provided. By“immunotherapy” is meant treatment of cell cycle protein relateddisorders with an antibody raised against a cell cycle protein. As usedherein, immunotherapy can be passive or active. Passive immunotherapy,as defined herein, is the passive transfer of antibody to a recipient(patient). Active immunization is the induction of antibody and/orT-cell responses in a recipient (patient). Induction of an immuneresponse can be the consequence of providing the recipient with an cellcycle protein antigen to which antibodies are raised. As appreciated byone of ordinary skill in the art, the cell cycle protein antigen may beprovided by injecting an cell cycle protein against which antibodies aredesired to be raised into a recipient, or contacting the recipient withan cell cycle protein nucleic acid, capable of expressing the cell cycleprotein antigen, under conditions for expression of the cell cycleprotein antigen.

[0222] In a preferred embodiment, a therapeutic compound is conjugatedto an antibody, preferably an cell cycle protein antibody. Thetherapeutic compound may be a cytotoxic agent. In this method, targetingthe cytotoxic agent to apoptotic cells or tumor tissue or cells, resultsin a reduction in the number of afflicted cells, thereby reducingsymptoms associated with apoptosis, cancer cell cycle protein relateddisorders. Cytotoxic agents are numerous and varied and include, but arenot limited to, cytotoxic drugs or toxins or active fragments of suchtoxins. Suitable toxins and their corresponding fragments includediptheria A chain, exotoxin A chain, ricin A chain, abrin A chain,curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents alsoinclude radiochemicals made by conjugating radioisotopes to antibodiesraised against cell cycle proteins, or binding of a radionuclide to achelating agent that has been covalently attached to the antibody.

[0223] In a preferred embodiment, cell cycle protein genes areadministered as DNA vaccines, either single nucleic acids orcombinations of cell cycle protein genes. Naked DNA vaccines aregenerally known in the art; see Brower, Nature Biotechnology16:1304-1305 (1998). Methods for the use of nucleic acids as DNAvaccines are well known to one of ordinary skill in the art, and includeplacing an cell cycle protein gene or portion of an cell cycle proteinnucleic acid under the control of a promoter for expression in apatient. The cell cycle protein gene used for DNA vaccines can encodefull-length cell cycle proteins, but more preferably encodes portions ofthe cell cycle proteins. including peptides derived from the cell cycleprotein. In a preferred embodiment a patient is immunized with a DNAvaccine comprising a plurality of nucleotide sequences derived from acell cycle protein gene. Similarly, it is possible to immunize a patientwith a plurality of cell cycle protein genes or portions thereof, asdefined herein. Without being bound by theory, following expression ofthe polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helperT-cells and antibodies are induced which recognize and destroy oreliminate cells expressing cell cycle proteins.

[0224] In a preferred embodiment, the DNA vaccines include a geneencoding an adjuvant molecule with the DNA vaccine. Such adjuvantmolecules include cytokines that increase the immunogenic response tothe cell cycle protein encoded by the DNA vaccine. Additional oralternative adjuvants are known to those of ordinary skill in the artand find use in the invention.

[0225] It is understood that the invention can be varied. All referencescited herein are expressly incorporated by reference in their entirety.Moreover, all sequences displayed, cited by reference or accessionnumber in the references are incorporated by reference herein.

We claim:
 1. A recombinant nucleic acid encoding a cell cycle proteincomprising a nucleic acid that hybridizes under high stringencyconditions to a sequence complementary to that set forth in FIG. 1 orFIG.
 3. 2. The recombinant nucleic acid of claim 1 wherein said proteinbinds to RIP3.
 3. The recombinant nucleic acid of claim 1 comprising anucleic acid sequence as set forth in FIG. 1 or FIG.
 3. 4. A recombinantnucleic acid encoding a cell cycle protein comprising a nucleic acidhaving at least 85% sequence identity to a sequence as set forth in FIG.1 or FIG.
 3. 5. A recombinant nucleic acid encoding an amino acidsequence as shown in FIG.
 2. 6. An expression vector comprising therecombinant nucleic acid according to any one of claims 1, 2, 3, 4, or5, operably linked to regulatory sequences recognized by a host celltransformed with the nucleic acid.
 7. A host cell comprising therecombinant nucleic acid according to any one of claims 1, 2, 3, 4, or5.
 8. A host cell comprising the vector of claim
 6. 9. A process forproducing a cell cycle protein comprising culturing the host cell ofclaim 8 under conditions suitable for expression of a cell cycleprotein.
 10. A process according to claim 9 further comprisingrecovering said cell cycle protein.
 11. A recombinant cell cycle proteinencoded by the nucleic acid of any of claims 1, 2, 3, 4, or
 5. 12. Arecombinant polypeptide comprising an amino acid sequence having atleast 80% sequence identity with the sequence set forth in FIG.
 2. 13.The recombinant polypeptide of claim 12 wherein said polypeptide bindsto RIP3.
 14. The recombinant polypeptide of claim 12 wherein saidsequence is set forth in FIG.
 2. 15. An isolated polypepbde whichspecifically binds to a cell cycle protein according to claim
 13. 16. Apolypeptide according to claim 15 that is an antibody.
 17. A polypeptideaccording to claim 16 wherein said antibody is a monoclonal antibody.18. The monoclonal antibody of claim 17 wherein said antibody reduces oreliminates the biological function of said cell cycle protein.
 19. Amethod for screening for a bioactive agent capable of binding to a cellcycle protein, said method comprising: a) combining a cell cycle proteinand a candidate bioactive agent; and b) determining the binding of saidcandidate bioactive agent to said cell cycle protein.
 20. A method forscreening for a bioactive agent capable of interfering with the bindingof a cell cycle protein and a RIP3 protein, said method comprising: a)combining a cell cycle protein, a candidate bioactive agent and a RIP3protein; and b) determining the binding of said cell cycle protein andsaid RIP3 protein.
 21. A method according to claim 20, wherein said cellcycle protein and said RIP3 protein are combined first.
 22. A method forscreening for a bioactive agent capable of modulating the activity ofcell cycle protein, said method comprising: a) adding a candidatebioactive agent to a cell comprising a recombinant nucleic acid encodinga cell cycle protein; and b) determining the effect of said candidatebioactive agent on said cell.
 23. A method according to claim 22,wherein a library of candidate bioactive agents is added to a pluralityof cells comprising a recombinant nucleic acid encoding a cell cycleprotein.